Modulators of B-lymphocyte activation, myosin-1F compositions and methods of use

ABSTRACT

The present invention provides compositions and methods for modulating B-lymphocyte activation. Nucleic acids encoding proteins and proteins so encoded which are capable of modulating B-lymphocyte activation are provided. Compositions and methods for the treatment of disorders related to dysfunction or dysregulation of B-lymphocyte activation are also provided. Prophylactics and methods for the prevention of such disorders are also provided. Also provided are compositions and methods for diagnostic and prognostic determination of such disorders. Further provided are assays for the identification of bioactive agents capable of modulating B-lymphocyte activation.

FIELD OF THE INVENTION

[0001] The present invention relates to B-lymphocyte activation andplatelet proliferation, and provides nucleic acids and proteins whichare capable of modulating B-lymphocyte activation and plateletproliferation. The invention concerns disorders related to thedysfunction and dysregulation of B-lymphocyte activation, as well asdisorders related to the dysfunction and dysregulation of plateletproliferation. These disorders include autoimmune diseases, acute andchronic inflammatory diseases, lymphomas, leukemias, and Wiskott-Aldrichsyndrome. The invention further concerns the immune response of a hostreceiving a transplant. The invention further concerns disorders relatedto the dysfunction or dysregulation of myosin-1F.

BACKGROUND OF THE INVENTION

[0002] The immune response comprises a cellular response and a humoralresponse. The cellular response is mediated largely by T lymphocytes(alternatively and equivalently referred to herein as T-cells), whilethe humoral response is mediated by B lymphocytes (alternatively andequivalently referred to herein as B-cells).

[0003] B-cells produce and secrete antibodies in response to thepresentation of antigen and MHC class II molecules on the surface ofantigen presenting cells Antigen presentation initiates B-cellactivation with the engagement of the B-cell receptor (BCR) at thecell's surface. Following engagement, the BCR relays signals that arepropagated through the cell's interior via signal transduction pathways.These signals lead to changes in B-cell gene expression and physiology,which underlie B-cell activation.

[0004] T-cells produce costimulatory molecules, including cytokines,that augment antibody production by B-cells during the humoral immuneresponse. Cytokines also play a role in modulating the activity ofT-cells themselves. Many T-cells act directly to engulf and destroycells or agents that they recognize by virtue of the cell surfaceproteins they possess. The engagement of cell surface receptors onT-cells results in the propagation of intracellular signals that provokechanges in T-cell gene expression and physiology, which underlie thecellular immune response.

[0005] Antigen recognition alone is usually not sufficient to initiate acomplete effector T or B-cell response. The generation of many B-cellresponses to antigen is dependent upon the interaction of B-cells withCD4+ helper T-cells directed against the same antigen. These helperT-cells express CD40L (CD154) which binds to the cell surface receptor,CD40, on resting B-cells. This interaction provides a criticalactivation signal to B-cells. Mutations in the CD40L lead to theX-linked immunodeficiency disorder hyper-IgM syndrome, which ischaracterized by low levels of IgA and IgG, normal to elevated levels ofIgM, absence of germinal center formation, and decreased immuneresponse. In addition, transgenic mice lacking CD40 exhibit reducedgraft rejection. (Zanelli et al., Nature Medicine, 6: 629-630, 2000;Schonbeck et al., Cell Mol Life Sci, 58:443, 2001).

[0006] Intercellular communication between different types oflymphocytes, as well as between lymphocytes and non-lymphocytes in thenormally functioning immune system is well known. Much of thiscommunication is mediated by cytokines and their cognate receptors.Cytokine-induced signals begin at the cell surface with a cytokinereceptor and are transmitted intracellularly via signal transductionpathways. Many types of cells produce cytokines, and cytokines caninduce a variety of responses in a variety of cell types, includinglymphocytes. The response to a cytokine can be context-dependent as wellas cell type specific.

[0007] Dysregulation of intercellular communication can perturblymphocyte activity and the regulation of immune responses. Suchdysregulation is believed to underlie certain autoimmune disease states,hyper-immune states, and immune-compromised states. Such dysfunction maybe cell autonomous or non-cell autonomous with respect to lymphocytes.

[0008] The activation of specific signaling pathways in lymphocytedetermines the quality, magnitude, and duration of immune responses. Inresponse to transplantation, in acute and chronic inflammatory diseases,and in autoimmune responses, it is these pathways that are responsiblefor the induction, maintenance and exacerbation of undesirablelymphocyte responses. Identification of these signaling pathways isdesirable in order to provide diagnostic and prognostic tools, as wellas therapeutic targets for modulating lymphocyte function in a varietyof disorders or abnormal physiological states. In addition, the abilityto modulate these pathways and suppress normal immune responses is oftendesirable, for example in the treatment of hosts receiving a transplant.

[0009] The cytoskeleton is a target of some signal transduction pathwaysand regulation of the actin cytoskeleton is an important point ofcontrol in the immune response. The migration of lymphocytes in responseto chemokines, the division of lymphocytes in response to cytokines andantigens, and the cellular shape changes associated with the developmentof plasma cells from pre B-cells, all involve changes in the actincytoskeleton.

[0010] Myosin proteins are important regulators of actin organization,as well as motor proteins which interact with actin filaments to mediateimportant cellular functions, e.g., vesicle trafficking.

[0011] Unconventional myosins make up a diverse group of multidomainactin-based motor proteins which have been implicated in the regulationof focal actin polymerization and the trafficking of actin andphospholipids along actin fibers. The class I myosins contain anN-terminal myosin head domain, comprising an ATP-binding motif and anactin binding site. The myosin head domain has ATPase activity andexhibits ATP-dependent actin binding activity. Following the myosin headdomain is an IQ domain(s), which mediates binding to the calcium-bindingprotein “calmodulin”. Following the IQ domain are three domains, denotedTH1, TH2 and TH3 (Crozet et al., Genomics, 40: 332-341, 1997).

[0012] The TH1 domain is rich in basic residues and mediates myosin-1binding to phospholipds. The TH2 domain is enriched in glycine, prolineand alanine, and may mediate ATP-independent binding to actin. TheC-terminal TH3 domain is an SH3 domain, which mediates protein-proteininteractions (Crozet et al., supra).

SUMMARY OF THE INVENTION

[0013] The present invention provides compositions and methods formodulating B-lymphocyte and platelet activation. Compositions andmethods for the treatment of disorders related to the dysfunction anddysregulation of B-lymphocyte and platelet activation are also provided.Prophylactics and methods for the prevention of such disorders are alsoprovided. Also provided are compositions and methods for the diagnosticand prognostic determination of such disorders. Further provided areassays for the identification of bioactive agents capable of modulatingB-lymphocyte and platelet activation.

[0014] Accordingly, in one aspect, the invention provides myosin-1Fnucleic acids which are capable of modulating B-lymphocyte and plateletactivation. In another aspect, the invention provides myosin-1F proteinscapable of modulating B-lymphocyte and platelet activation.

[0015] In a preferred embodiment, the invention provides myosin-1Fnucleic acids which encode myosin-1F proteins.

[0016] In a preferred embodiment, the present invention providesmyosin-1F proteins which can bind to one or more myosin-1F bindingpartners selected from the group consisting of BLNK, WASP, WASP-IP,Bee1p, Vrp1p, calmodulin, Arp2/3 complex, Acan125, ATP and actin.

[0017] In a preferred embodiment, a myosin-1F protein provided herein iscapable of modulating B-cell receptor (BCR)-induced expression of CD69in a B-lymphocyte.

[0018] In an especially preferred embodiment, a myosin-1F proteinprovided herein is capable of modulating BCR-induced expression of CD69in a B-lymphocyte, but does not modulate T-cell receptor (TCR)-inducedCD69 expression in a T-lymphocyte.

[0019] In a preferred embodiment, a myosin-1F protein provided herein iscapable of modulating BCR-induced activation of the immunoglobulin heavychain gene (IgH) promoter in a B-lymphocyte. In an especially preferredembodiment, such a myosin-1F protein does not modulate TCR-inducedT-cell activation.

[0020] In a preferred embodiment, a myosin-1F protein provided herein iscapable of modulating BCR-induced immunoglobulin production in aB-lymphocyte. In an especially preferred embodiment, such a myosin-1Fprotein does not modulate TCR-induced T-cell activation.

[0021] In a preferred embodiment, a myosin-1F protein provided herein iscapable of modulating intracellular calcium increase induced by antigenreceptor activation in B-lymphocytes. In an especially preferredembodiment, such a myosin-1F protein does not modulate TCR-inducedT-cell activation.

[0022] In a preferred embodiment, a myosin-1F protein provided herein iscapable of modulating antigen receptor-induced calcineurin activity inB-lymphocytes. In an especially preferred embodiment, such a myosin-1Fprotein does not modulate TCR-induced T-cell activation.

[0023] In a preferred embodiment, a myosin-1F protein provided herein iscapable of modulating the level of surface Ig, preferably surface IgMexpression, on a resting B-lymphocyte.

[0024] In a preferred embodiment, a myosin-1F protein provided hereincomprises the consensus phosphorylation site sequence GRSESINV occurringupstream of a conserved DALAK sequence, as in SEQ ID NO:2.

[0025] In a preferred embodiment, a myosin-1F protein provided herein isa substrate for phosphorylation by a protein kinase, preferably a PAK,as is known for myosin-1F homologs.

[0026] In a preferred embodiment, a myosin-1F nucleic acid providedherein is expressed in the following tissues and cells, in decreasingorder of abundance of expression: lung, bone marrow, peripheral bloodmononuclear cells, heart, spleen, placenta, HL-60 cells, liver, smallintestine, BJAB cells, colon, trachea, uterus, adrenal gland, thymus,skeletal muscle, prostate, salivary gland, testis, thyroid, kidney,pancreas, whole brain, MCF-7 cells, Huh7 cells, cerebellum, phoenix Acells, and Jurkat cells.

[0027] The present invention also provides isolated polypeptides whichspecifically bind to a myosin-1F protein. In one embodiment, thepolypeptide is an antibody. In a preferred embodiment, the polypeptideis a monoclonal antibody. In one embodiment, the monoclonal antibody iscapable of reducing or eliminating the activity of myosin-1F. In anotherembodiment, the monoclonal antibody is capable of increasing orenhancing the activity of myosin-1F.

[0028] Also provided herein are methods of screening for a bioactiveagent capable of binding to a myosin-1F protein. The methods comprisecombining a myosin-1F protein and a candidate bioactive agent anddetermining the binding of candidate agent to myosin-1F protein. In oneembodiment, the method involves identifying the candidate agent.

[0029] Also provided herein are methods of screening for a bioactiveagent capable of interfering with the binding of a myosin-1F protein.The methods comprise combining a candidate bioactive agent, a myosin-1Fprotein, and a myosin-1F binding partner which will bind to myosin-1F inthe absence of candidate agent, and determining the binding of myosin-1Fto binding partner in the presence of candidate bioactive agent. In apreferred embodiment, the myosin-1F binding partner is selected from thegroup consisting of Wiskott-Aldrich syndrome protein-interacting protein(WASP), WASP-interacting protein (WASP-IP), BLNK, Bee1p, Vrp1p,calmodulin, Arp2/3 complex, Acan125, ATP and actin. In a preferredembodiment, the method involves determining the binding of myosin-1F tobinding partner in the presence and absence of candidate bioactiveagent. In one embodiment, myosin-1F and myosin-1F binding partner arecombined first. In one embodiment, the method involves identifying thecandidate agent.

[0030] Also provided herein are methods of screening for a bioactiveagent capable of increasing the binding of a myosin-1F protein. Themethods comprise combining a candidate bioactive agent, a myosin-1Fprotein, and a myosin-1F binding partner which will bind to myosin-1F inthe absence of candidate agent, and determining the binding of myosin-1Fto binding partner in the presence of candidate bioactive agent. In apreferred embodiment, the myosin-1F binding partner is selected from thegroup consisting of WASP, BLNK, WASP-IP, Bee1p, Vrp1p, calmodulin,Arp2/3 complex, Acan125, ATP and actin. In a preferred embodiment, themethod involves determining the binding of myosin-1F to binding partnerin the presence and absence of candidate bioactive agent. In oneembodiment, myosin-1F and myosin-1F binding partner are combined first.In one embodiment, the method involves identifying the candidate agent.

[0031] Also provided herein are methods of screening for a bioactiveagent capable of modulating the activity of a myosin-1F protein. In apreferred embodiment, the method comprises contacting a candidatebioactive agent to a cell comprising a recombinant myosin-1F nucleicacid and expressing a myosin-1F protein. In a preferred embodiment, themethod comprises contacting a library of candidate bioactive agents to aplurality of cells comprising a recombinant myosin-1F nucleic acid andexpressing a myosin-1F protein. In a preferred embodiment, the methodcomprises determining ATPase activity. In another preferred embodiment,the method comprises determining actin polymerization. In anotherpreferred embodiment, the method comprises determining intracellularcalcium concentration.

[0032] Also provided herein are methods of screening for a bioactiveagent capable of modulating B-lymphocyte activation.

[0033] In a preferred embodiment, the methods comprise determining theability of a candidate agent to bind to myosin-1F.

[0034] In a preferred embodiment, the methods comprise detecting bindingof candidate agent to myosin-1F protein, contacting the candidatebioactive agent to a B-lymphocyte, and determining lymphocyte activationin the presence of said candidate agent. In a preferred embodiment,lymphocyte activation in the presence and absence of candidate agent isdetermined. In a preferred embodiment, an agent that normally induceslymphocyte activation is used. Lymphocyte activation is measured in thepresence, and optionally, the absence of candidate agent followingcontact with the agent that normally induces lymphocyte activation. Apreferred activation agent for use with B lymphocytes is anti-IgMantibody.

[0035] Bioactive agents that inhibit B-lymphocyte activation in theseassays are useful as immunosuppressants.

[0036] In another preferred embodiment, the methods comprise determiningthe ability of a candidate agent to modulate the binding of myosin-1F toa myosin-1F binding partner.

[0037] In a preferred embodiment, the methods comprise detectingmodulation of the binding of myosin-1F protein to a myosin-1F bindingpartner in the presence of candidate agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining lymphocyte activationin the presence of said candidate agent. In a preferred embodiment,lymphocyte activation in the presence and absence of candidate agent isdetermined. In a preferred embodiment, an agent that normally-induceslymphocyte activation is used. Lymphocyte activation is measured in thepresence, and optionally, the absence of candidate agent followingcontact with the agent that normally induces lymphocyte activation. Apreferred activation agent for use with B lymphocytes is anti-IgMantibody. In an especially preferred embodiment, the myosin-1F bindingpartner used in these methods is selected from the group consisting ofWASP, BLNK, WASP-IP, Bee1p, Vrp1p, calmodulin, Arp2/3 complex, Acan125,ATP and actin.

[0038] Bioactive agents that inhibit B-lymphocyte activation in theseassays are useful as immunosuppressants.

[0039] In another preferred embodiment, the methods comprise determiningthe ability of a candidate bioactive agent to modulate the activity of amyosin-1F protein.

[0040] In a preferred embodiment, the methods comprise detectingmodulation of ATPase activity, or modulation of actin binding activity,or modulation of actin polymerization inducing activity of myosin-1F inthe presence of candidate bioactive agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining lymphocyte activationin the presence of said candidate agent. In a preferred embodiment,lymphocyte activation in the presence and absence of candidate agent isdetermined. In a preferred embodiment, an agent that normally induceslymphocyte activation is used. Lymphocyte activation is measured in thepresence, and optionally, the absence of candidate agent followingcontact with the agent that normally induces lymphocyte activation. Apreferred activation agent for use with B lymphocytes is anti-IgMantibody.

[0041] Bioactive agents that inhibit lymphocyte activation in theseassays are useful as immunosuppressants.

[0042] In a preferred embodiment, the methods comprise detecting bindingof candidate agent to myosin-1F protein, contacting the candidatebioactive agent to a B-lymphocyte, and determining CD23 expression inthe presence of said candidate agent. In a preferred embodiment, CD23expression in the presence and absence of candidate agent is determined.In a preferred embodiment, an agent that normally induces CD23expression is used. Lymphocyte activation is measured in the presence,and optionally, the absence of candidate agent following contact withthe agent that normally induces CD23 expression. Preferred CD23 inducingagents are IL-4, CD40L, and the combination of IL-4 and CD40L.

[0043] Bioactive agents that inhibit CD23 induction in these assays areuseful as immunosuppressants.

[0044] In a preferred embodiment, the methods comprise detectingmodulation of the binding of myosin-1F protein to a myosin-1F bindingpartner in the presence of candidate agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining CD23 expression inthe presence of said candidate agent. In a preferred embodiment, CD23expression in the presence and absence of candidate agent is determined.In a preferred embodiment, an agent that normally induces CD23expression is used. Lymphocyte activation is measured in the presence,and optionally, the absence of candidate agent following contact withthe agent that normally induces CD23 expression. Preferred CD23 inducingagents are IL-4, CD40L, and the combination of IL-4 and CD40L.

[0045] In an especially preferred embodiment, the myosin-1F bindingpartner used in these methods is selected from the group consisting ofWASP, BLNK, WASP-IP, Bee1p, Vrp1p, calmodulin, Arp2/3 complex, Acan125,ATP and actin.

[0046] Bioactive agents that inhibit CD23 induction in these assays areuseful as immunosuppressants.

[0047] In a preferred embodiment, the methods comprise detectingmodulation of ATPase activity, or modulation of actin binding activity,or modulation of actin polymerization inducing activity of myosin-1F inthe presence of candidate bioactive agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining CD23 expression inthe presence of said candidate agent. In a preferred embodiment, CD23expression in the presence and absence of candidate agent is determined.In a preferred embodiment, an agent that normally induces CD23expression is used. Lymphocyte activation is measured in the presence,and optionally, the absence of candidate agent following contact withthe agent that normally induces CD23 expression. Preferred CD23 inducingagents are IL-4, CD40L, and the combination of IL-4 and CD40L.

[0048] Bioactive agents that inhibit CD23 induction in these assays areuseful as immunosuppressants.

[0049] In a preferred embodiment, the methods comprise detecting bindingof candidate agent to myosin-1F protein, contacting the candidatebioactive agent to a resting B-lymphocyte, and determining the level ofsurface Ig expression, preferably surface IgM expression, in the cell.Particularly preferred are Ramos cells and primary B-cells.

[0050] In a preferred embodiment, the methods comprise detectingmodulation of the binding of myosin-1F protein to a myosin-1F bindingpartner in the presence of candidate agent, contacting the candidatebioactive agent to a resting B-lymphocyte, and determining the level ofsurface Ig expression, preferably surface IgM expression, in the cell.Particularly preferred are Ramos cells and primary B-cells.

[0051] In a preferred embodiment, the methods comprise detectingmodulation of ATPase activity, or modulation of actin binding activity,or modulation of actin polymerization inducing activity of myosin-1F inthe presence of candidate bioactive agent, contacting the candidatebioactive agent to a resting B-lymphocyte, and determining the level ofsurface Ig expression, preferably surface IgM expression, in the cell.Particularly preferred are Ramos cells and primary B-cells.

[0052] Agents that decrease surface Ig expression in resting B-cells areparticularly preferred, and are useful as immunosuppressants.

[0053] In a preferred embodiment, candidate bioactive agents used inthese assays are small molecule chemical compounds, from about 100 toabout 1500, more preferably about 100 to about 1200, more preferablyabout 100 to about 1000, more preferably about 200 to about 500 daltons.

[0054] In a preferred embodiment, a library of candidate bioactiveagents is contacted to myosin-1F protein.

[0055] In a preferred embodiment, determining lymphocyte activation inthe methods herein comprises determining the level of expression of asurface marker which is associated with activation of the lymphocyte, inthe presence of candidate agent. In a preferred embodiment, the surfacemarker is selected from the group consisting of CD23, CD69, CD80, andCD86. In an especially preferred embodiment, the surface marker is CD69or CD23.

[0056] In another preferred embodiment, determining lymphocyteactivation in the methods herein comprises determining the level ofactivity of a promoter in the presence of candidate agent, whichactivity correlates with lymphocyte activation in the absence ofcandidate agent. In a preferred embodiment the promoter is anNFAT-responsive promoter, such as the IL-2 promoter. In anotherpreferred embodiment, the promoter is the immunoglobulin heavy chaingene promoter.

[0057] In another preferred embodiment, determining lymphocyteactivation in the methods herein comprises determining the intracellularcalcium concentration in the presence of candidate agent. In a preferredembodiment, the intracellular calcium concentration is determined in thepresence and absence of candidate agent. Preferably, calciumconcentration is determined using a calcium sensitive dye.

[0058] In a preferred embodiment, determining lymphocyte activation,including CD23 induction, is done using a FACS machine. In a preferredembodiment, lymphocytes are sorted by FACS. A FACS machine may be usedto determine the level of expression of a surface marker orintracellular marker which normally correlates with lymphocyteactivation, or the level of activity of a promoter which normallycorrelates with lymphocyte activation, or the intracellular calciumlevel, or other indicators of lymphocyte activation, including thosediscussed herein. Sorting of lymphocytes may be done on these bases.

[0059] Also provided herein are methods for inhibiting immunoglobulinproduction in a B-cell. In one embodiment, the methods compriseintroducing into a B cell an immunosuppressant identified by the methodsprovided herein. In one embodiment, the methods comprise introducinginto a B-cell a modulator of myosin-1F activity.

[0060] Also provided herein are methods for modulating lymphocyteactivation in a patient having a lymphocyte activation disorder,comprising administering to a patient having a lymphocyte activationdisorder a medicament comprising a modulator of myosin-1F proteinactivity.

[0061] Also provided herein are methods for inhibiting B cell activationin a patient having an autoimmune disease, comprising administering to apatient having an autoimmune disease a medicament comprising a modulatorof myosin-1F activity.

[0062] Also provided herein are methods for inhibiting B cell activationin a patient having an autoimmune disease, comprising administering to apatient having an autoimmune disease a medicament comprising animmunosuppressant obtained by the screening methods provided herein.

[0063] Also provided herein are methods for inhibiting immunoglobulinproduction in a patient having an autoimmune disease, comprisingadministering to a patient having an autoimmune disease a medicamentcomprising a modulator of myosin-1F activity.

[0064] Also provided herein are methods for inhibiting immunoglobulinproduction in a patient having an autoimmune disease, comprisingadministering to a patient having an autoimmune disease a medicamentcomprising an immunosuppressant identified by the methods providedherein.

[0065] Also provided herein are methods for prolonging the survival of agraft in a mammalian host, comprising administering to a mammalian hostreceiving a graft a medicament comprising a modulator of myosin-1Factivity.

[0066] Also provided herein are methods for prolonging the survival of agraft in a mammalian host, comprising administering to a mammalian hostreceiving a graft a medicament comprising an immunosuppressantidentified by the methods provided herein.

[0067] In a preferred embodiment, provided herein are small moleculechemical compositions useful for the prevention and treatment of acuteinflammatory disorders, chronic inflammatory disorders, autoimmunedisorders, and transplant rejection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0068]FIG. 1 shows nucleotide sequence of human myosin-1F, SEQ ID NO:1.

[0069]FIG. 2 shows amino acid sequence of human myosin-1F, SEQ ID NO:2.

[0070]FIG. 3 shows the results from experiments in which a myosin-1Ffragment, consisting of an IQ domain and a portion of the tail domain(“Myosin 1F Hit”) (amino acids 617-1098 of SEQ ID NO:2; nucleic acid SEQID NO:5), and a longer isoform of myosin-1F (myosin-1F long; SEQ IDNO:4) were expressed in the BJAB (B-cell) cell line and the Jurkat(T-cell) cell line, and BCR- or TCR-induced endogenous CD69 expressionwas assayed using a FACS machine. The results demonstrate that the shortmyosin-1F fragment inhibits BCR-induced lymphocyte activation but notTCR-induced lymphocyte activation, as measured by CD69 expression. Theresults also demonstrate that overexpression of the longer myosin-1Fisoform does not affect BCR-induced or TCR-induced CD69 expression inBJAB and Jurkat cells.

[0071]FIG. 4 shows the results from experiments in which a myosin-1Ffragment, consisting of an IQ domain and a portion of the tail domain(“Myosin 1F Hit”) (amino acids 617-1098 of SEQ ID NO:2; nucleic acid SEQID NO:5), and a longer isoform of myosin-1F (myosin-1F long; SEQ IDNO:4) were expressed in the BJAB cell line and the Jurkat cell line, andBCR- or TCR-induced calcium increase was detected using a fluorescentcalcium dye which was measured by a FACS machine. The resultsdemonstrate that the short myosin-1F fragment inhibits intracellularcalcium increase in BJAB cells but not Jurkat cells. The results furthershow that the longer myosin-1F isoform does not affect BCR-induced orTCR-induced calcium intracellular increase in BJAB and Jurkat cells.

[0072]FIG. 5 is a table of human autoimmune diseases.

[0073]FIG. 6 shows an alignment of ATP binding site sequences betweendifferent myosin isoforms.

[0074]FIG. 7 shows an expression profile of myosin-1F mRNA in blood cellsubsets in the resting and activated states.

[0075]FIG. 8 shows the nucleotide sequence of a truncated myosin-1Fisoform (myosin-1F long, above) (SEQ ID NO:3).

[0076]FIG. 9 shows the amino acid sequence of a truncated humanmyosin-1F protein (myosin-1F long above) (SEQ ID NO:4) and itsrelationship to myosin-1F hit and full length myosin-1F.

[0077]FIG. 10 shows the nucleotide sequence of myosin-1F hit (SEQ IDNO:5).

DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention provides compositions and methods formodulating B-lymphocyte activation and platelet activation. Compositionsand methods for the treatment of disorders related to the dysfunctionand dysregulation of B-lymphocyte activation and platelet activation arealso provided. Prophylactics and methods for the prevention of suchdisorders are also provided. Also provided are compositions and methodsfor the diagnostic and prognostic determination of such disorders.Further provided are assays for the identification of bioactive agentscapable of modulating B-lymphocyte activation and platelet activation.

[0079] In accordance with these objectives, in one aspect, the inventionprovides myosin-1F nucleic acids which are capable of modulatingB-lymphocyte and platelet activation. Also in accordance with theseobjectives, in another aspect, the invention provides myosin-1F proteinscapable of modulating B-lymphocyte and platelet activation.

[0080] A myosin-1F protein of the present invention may be identified inseveral ways. “Protein” in this sense includes proteins, polypeptides,and peptides. A myosin-1F protein may be identified by its ability tobind to myosin-1F binding partners as described herein. A myosin-1Fprotein may be identified by its ability to bind ATP, its ability tobind to actin in an ATP-dependent manner, its ability to bind to actinin an ATP-independent manner, its ability to bind to phospholipids, itsability to bind to calmodulin, its ATPase activity, and combinations ofthese properties. A myosin-1F protein may also be identified by aminoacid sequence identity or similarity to SEQ ID NO:2, or by the sequenceidentity or similarity of its encoding nucleic acid to SEQ ID NO:1, morepreferably the open reading frame (nucleotides 41-3337) of SEQ ID NO:1.

[0081] Myosin-1F nucleic acids and proteins may initially be identifiedby sequence identity or similarity to SEQ ID NOs:1 and 2, as furtherdescribed below. In a preferred embodiment, myosin-1F nucleic acids andmyosin-1F proteins have sequence identity or similarity to the sequencesprovided herein and one or more myosin-1F bioactivities describedherein. Such sequence identity or similarity can be based upon theoverall nucleic acid or amino acid sequence.

[0082] In a preferred embodiment, a myosin-1F protein provided hereincomprises an amino acid sequence having at least about 85%, morepreferably at least about 90%, more preferably at least about 95%, morepreferably at least about 98% identity to the amino acid sequence setforth in SEQ ID NO:2. In a preferred embodiment, the myosin-1F proteincomprises the amino acid sequence set forth in SEQ ID NO:2. Preferably,the myosin-1F protein also possesses one or more myosin-1F bioactivitiesdescribed herein.

[0083] Myosin-1F protein having the amino acid sequence of SEQ ID NO:2has been previously identified (Crozet et al., Genomics, 40:332-341,1997). Myosin-1F is a member of the unconventional myosin I gene family,and comprises a number of conserved domains typically found in familymembers. Particularly, myosin-1F comprises an N-terminal myosin headdomain having an ATP-binding site and actin binding site. The myosinhead domain has ATPase activity and can bind to actin in anATP-dependent manner. Following the myosin head domain, the “neckregion” of the protein comprises a single IQ domain which binds to thecalcium-binding protein “calmodulin”. Following the single IQ domain arelocated a basic domain (TH1) which binds phospholipids, aglycine/proline/alanine-rich region (TH2) which binds actin in anATP-independent manner, and a C-terminal SH3 domain (Crozet et al.,supra). In addition, as with all vertebrate unconventional myosin Iproteins, the myosin head domain lacks the conserved “TEDS rule”phosphorylation site found in protozoan myosins (Novak et al., Mol.Biol. of Cell, 9:75-88, 1998) and possesses a variation of thissequence.

[0084] Fragments are included in the definition of myosin-1F proteinsherein. In a preferred embodiment, a myosin-1F protein provided hereincomprises an amino acid sequence having at least about 85%, morepreferably at least about 90%, more preferably at least about 95%, morepreferably at least about 98% identity to a portion of the amino acidsequence set forth in SEQ ID NO:2. In a preferred embodiment, themyosin-1F protein comprises a portion of the amino acid sequence setforth in SEQ ID NO:2. Portion, or fragment, in this sense includessequences from at least 2 amino acids up to the full length sequence inSEQ ID NO:2 minus one amino acid at either the N- or C-terminus.

[0085] In a preferred embodiment, such a myosin-1F protein comprises theamino acid sequence set forth in SEQ ID NO:4.

[0086] In an especially preferred embodiment, such a myosin-1F proteincomprises a myosin head domain which can bind to ATP, possesses ATPaseactivity, and which can bind to actin, preferably in an ATP-dependentmanner. In a preferred embodiment, such a myosin-1F protein comprises anamino acid sequence having at least about 85%, more preferably at leastabout 90%, more preferably at least about 95%, more preferably at leastabout 98% identity to the amino acid sequence set forth by residues19-677, 1-677, 12-691, or 1-691 in SEQ ID NO:2. In an especiallypreferred embodiment, such a myosin-1F protein comprises the amino acidsequence set forth by residues 19-677, 1-677, 12-691, or 1-691 in SEQ IDNO:2.

[0087] In a preferred embodiment, such a myosin-1F protein comprises apartial myosin head domain. In a preferred embodiment, such a myosin-1Fprotein comprises an amino acid sequence having at least about 85%, morepreferably at least about 90%, more preferably at least about 95%, morepreferably at least about 98% identity to the amino acid sequence setforth by residues 260-677 or 260-691 in SEQ ID NO:2. In an especiallypreferred embodiment, such a myosin-1F protein comprises the amino acidsequence set forth by residues 260-677 or 260-691 in SEQ ID NO:2.

[0088] In another preferred embodiment, such a myosin-1F proteincomprises a partial myosin head domain, an IQ domain, and a tail domain.In a preferred embodiment, such a myosin-1F protein comprises an aminoacid sequence having at least about 85%, more preferably at least about90%, more preferably at least about 95%, more preferably at least about98% identity to the amino acid sequence set forth by residues 260-1098in SEQ ID NO:2. In an especially preferred embodiment, such a myosin-1Fprotein comprises the amino acid sequence set forth by residues 260-1098in SEQ ID NO:2. In another especially preferred embodiment, such amyosin-1F protein comprises the amino acid sequence set forth by SEQ IDNO:4.

[0089] In another especially preferred embodiment, such a myosin-1Fprotein comprises a tail domain, which comprises a TH1 domain, a TH2domain, and an SH3 domain, each of which is further described herein. Ina preferred embodiment, such a myosin-1F protein comprises an amino acidsequence having at least about 85%, more preferably at least about 90%,more preferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 718-1098 inSEQ ID NO:2. In an especially preferred embodiment, such a myosin-1Fprotein comprises the amino acid sequence set forth by residues 718-1098in SEQ ID NO:2.

[0090] In another especially preferred embodiment, such a myosin-1Fprotein comprises an IQ domain, as further described herein. In apreferred embodiment, such a myosin-1F protein comprises an amino acidsequence having at least about 85%, more preferably at least about 90%,more preferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 695-717 in SEQID NO:2. In an especially preferred embodiment, such a myosin-1F proteincomprises the amino acid sequence set forth by residues 695-717 in SEQID NO:2.

[0091] In another especially preferred embodiment, such a myosin-1Fprotein comprises an IQ domain and a tail domain. In a preferredembodiment, such a myosin-1F protein comprises an amino acid sequencehaving at least about 85%, more preferably at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 695-1098 inSEQ ID NO:2. In an especially preferred embodiment, such a myosin-1Fprotein comprises the amino acid sequence set forth by residues 695-1098in SEQ ID NO:2.

[0092] In another especially preferred embodiment, such a myosin-1Fprotein comprises an IQ domain and a tail domain. In a preferredembodiment, such a myosin-1F protein comprises an amino acid sequencehaving at least about 85%, more preferably at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 617-1098 inSEQ ID NO:2. In an especially preferred embodiment, such a myosin-1Fprotein comprises the amino acid sequence set forth by residues 617-1098in SEQ ID NO:2.

[0093] In another especially preferred embodiment, such a myosin-1Fprotein comprises a myosin head domain and an IQ domain. In a preferredembodiment, such a myosin-1F protein comprises an amino acid sequencehaving at least about 85%, more preferably at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 1-717, 12-717or 19-717 in SEQ ID NO:2. In an especially preferred embodiment, such amyosin-1F protein comprises the amino acid sequence set forth byresidues 1-717, 12-717 or 19-717 in SEQ ID NO:2.

[0094] In another preferred embodiment, a myosin-1F protein providedherein consists essentially of an amino acid sequence having at leastabout 85%, more preferably at least about 90%, more preferably at leastabout 95%, more preferably at least about 98% identity to a portion ofthe amino acid sequence set forth in SEQ ID NO:2. In a preferredembodiment, the myosin-1F protein consists essentially of a portion ofthe amino acid sequence set forth in SEQ ID NO:2.

[0095] In another preferred embodiment, a myosin-1F protein providedherein consists essentially of the amino acid sequence set forth in SEQID NO:4.

[0096] In an especially preferred embodiment, such a myosin-1F proteinconsists essentially of a myosin head domain which can bind to ATP,possesses ATPase activity, and which can bind to actin, preferably in anATP-dependent manner. In a preferred embodiment, such a myosin-1Fprotein consists essentially of an amino acid sequence having at leastabout 85%, more preferably at least about 90%, more preferably at leastabout 95%, more preferably at least about 98% identity to the amino acidsequence set forth by residues 19-677, 1-677, 12-691, or 1-691 in SEQ IDNO:2. In an especially preferred embodiment, such a myosin-1F proteinconsists essentially of the amino acid sequence set forth by residues19-677, 1-677, 12-691, or 1-691 in SEQ ID NO:2.

[0097] In a preferred embodiment, such a myosin-1F protein consistsessentially of a partial myosin head domain. In a preferred embodiment,such a myosin-1F protein consists essentially of an amino acid sequencehaving at least about 85%, more preferably at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 260-677 or260-691 in SEQ ID NO:2. In an especially preferred embodiment, such amyosin-1F protein consists essentially of the amino acid sequence setforth by residues 260-677 or 260-691 in SEQ ID NO:2.

[0098] In another preferred embodiment, such a myosin-1F proteinconsists essentially of a partial myosin head domain, an IQ domain, anda tail domain. In a preferred embodiment, such a myosin-1F proteinconsists essentially of an amino acid sequence having at least about85%, more preferably at least about 90%, more preferably at least about95%, more preferably at least about 98% identity to the amino acidsequence set forth by residues 260-1098 in SEQ ID NO:2. In an especiallypreferred embodiment, such a myosin-1F protein consists essentially ofthe amino acid sequence set forth by residues 260-1098 in SEQ ID NO:2.In another especially preferred embodiment, such a myosin-1F proteinconsists essentially of the amino acid sequence set forth by SEQ IDNO:4.

[0099] In another preferred embodiment, such a myosin-1F proteinconsists essentially of a tail domain, which comprises a TH1 domain, aTH2 domain, and an SH3 domain. In a preferred embodiment, such amyosin-1F protein consists essentially of an amino acid sequence havingat least about 85%, more preferably at least about 90%, more preferablyat least about 95%, more preferably at least about 98% identity to theamino acid sequence set forth by residues 718-1098 in SEQ ID NO:2. In anespecially preferred embodiment, such a myosin-1F protein consistsessentially of the amino acid sequence set forth by residues 718-1098 inSEQ ID NO:2.

[0100] In another preferred embodiment, such a myosin-1F proteinconsists essentially of an IQ domain and a tail domain which comprises aTH1 domain, a TH2 domain, and an SH3 domain. In a preferred embodiment,such a myosin-1F protein consists essentially of an amino acid sequencehaving at least about 85%, more preferably at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 695-1098 inSEQ ID NO:2. In an especially preferred embodiment, such a myosin-1Fprotein consists essentially of the amino acid sequence set forth byresidues 695-1098 in SEQ ID NO:2.

[0101] In another preferred embodiment, such a myosin-1F proteinconsists essentially of an IQ domain and a tail domain which comprises aTH1 domain, a TH2 domain, and an SH3 domain. In a preferred embodiment,such a myosin-1F protein consists essentially of an amino acid sequencehaving at least about 85%, more preferably at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to the amino acid sequence set forth by residues 617-1098 inSEQ ID NO:2. In an especially preferred embodiment, such a myosin-1Fprotein consists essentially of the amino acid sequence set forth byresidues 617-1098 in SEQ ID NO:2.

[0102] In another preferred embodiment, such a myosin-1F proteinconsists essentially of a myosin head domain and an IQ domain. In apreferred embodiment, such a myosin-1F protein consists essentially ofan amino acid sequence having at least about 85%, more preferably atleast about 90%, more preferably at least about 95%, more preferably atleast about 98% identity to the amino acid sequence set forth byresidues 1-717, 12-717 or 19-717 in SEQ ID NO:2. In an especiallypreferred embodiment, such a myosin-1F protein consists essentially ofthe amino acid sequence set forth by residues 1-717,12-717 or 19-717 inSEQ ID NO:2.

[0103] In another preferred embodiment, the such a myosin-1F proteinconsists essentially of a portion of a tail domain, particularly a TH2domain and an SH3 domain.

[0104] In one aspect, the present invention provides myosin-1F nucleicacids, including myosin-1F nucleic acids encoding myosin-1F proteins.

[0105] In the case of a myosin-1F nucleic acid encoding a myosin-1Fprotein, the overall sequence identity of the nucleic acid sequence iscommensurate with amino acid sequence identity but takes into accountthe degeneracy in the genetic code and codon bias of differentorganisms. Accordingly, the nucleic acid sequence identity may be eitherlower or higher than that of the protein sequence. A myosin-1F nucleicacid of the present invention comprises a nucleic acid sequence thatpreferably has greater than about 75% identity to the nucleic acidsequence set forth in SEQ ID NO: 1, more preferably greater than about80%, particularly greater than about 85% and most preferably greaterthan 90%. In some embodiments the sequence identity will be as high asabout 93 to 95 or 98%.

[0106] In a preferred embodiment, a myosin-1F nucleic acid encodes amyosin-1F protein. As will be appreciated by those in the art, due tothe degeneracy of the genetic code, an extremely large number of nucleicacids may be made, all of which encode the myosin-1F proteins of thepresent invention. Thus, having identified a particular amino acidsequence, those skilled in the art could make any number of differentnucleic acids, by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the myosin-1Fprotein.

[0107] In a preferred embodiment, the myosin-1F nucleic acid comprises anucleic acid sequence having at least about 85%, more preferably atleast about 90%, more preferably at least about 95%, more preferably atleast about 98% identity to the nucleic acid sequence set forth in SEQID NO:1, more preferably the open reading frame set forth in SEQ ID NO:1from nucleotides 41-3337. In a preferred embodiment, the myosin-1Fnucleic acid comprises the nucleic acid sequence set forth in SEQ IDNO:1, more preferably the open reading frame set forth in SEQ ID NO:1from nucleotides 41-3337. In a preferred embodiment, the myosin-1Fnucleic acid encodes a myosin-1F protein.

[0108] In a preferred embodiment, the myosin-1F nucleic acid comprises anucleic acid sequence having at least about 85%, more preferably atleast about 90%, more preferably at least about 95%, more preferably atleast about 98% identity to the sequence set forth by nucleotides1891-3558 in SEQ ID NO:1. In a preferred embodiment, the myosin-1Fnucleic acid comprises the sequence set forth by nucleotides 1891-3558in SEQ ID NO:1.

[0109] In a preferred embodiment, the myosin-1F nucleic acid comprisesthe sequence set forth by SEQ ID NO:3.

[0110] In a preferred embodiment, the myosin-1F nucleic acid comprisesthe sequence set forth by SEQ ID NO:5.

[0111] In a preferred embodiment, the present invention providesmyosin-1F proteins encoded by myosin-1F nucleic acids provided herein.

[0112] In a preferred embodiment, the present invention providesmyosin-1F nucleic acids encoding myosin-1F protein fragments describedherein.

[0113] In one embodiment, the myosin-1F nucleic acid is determinedthrough hybridization studies. Thus, for example, nucleic acids whichhybridize under high stringency conditions to the nucleic acid sequencesshown in SEQ ID NO:1 or their complements, or fragments thereof or theircomplements, are considered myosin-1F nucleic acids. High stringencyconditions are known in the art; see for example Sambrook et al.,Molecular Cloning, A Laboratory Manual, 3rd edition, 2001, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.; and Short Protocols in MolecularBiology, ed. Ausubel, et al., both of which are hereby incorporated byreference. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, “Overview of principles of hybridization and the strategy ofnucleic acid assays” (1993). Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH and nucleicacid concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.

[0114] In another embodiment, less stringent hybridization conditionsare used; for example, moderate or low stringency conditions may beused, as are known in the art; see Sambrook et al., Molecular Cloning, ALaboratory Manual, 3rd edition, 2001, Cold Spring Harbor Press, ColdSpring Harbor, N.Y.; and Tijssen, supra.

[0115] Also provided herein are myosin-1F antisense nucleic acids whichwill hybridize under high stringency conditions to a myosin-1F nucleicacid comprising the nucleic acid sequence set forth in SEQ ID NO: 1. Ina preferred embodiment, the myosin-1F antisense nucleic acid inhibitsexpression of myosin-1F protein. In a preferred embodiment, themyosin-1F antisense nucleic acid inhibits myosin-1F protein activity.

[0116] As is known in the art, a number of different programs can beused to identify whether a protein (or nucleic acid as discussed below)has sequence identity or similarity to a known sequence. Sequenceidentity and/or similarity is determined using standard techniques knownin the art, including, but not limited to, the local sequence identityalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thesequence identity alignment algorithm of Needleman & Wunsch, J. Mol.Biool. 48:443 (1970), by the search for similarity method of Pearson &Lipman, PNAS USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387-395 (1984), preferably using the defaultsettings, or by inspection. Preferably, percent identity is calculatedby FastDB based upon the following parameters: mismatch penalty of 1;gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30,“Current Methods in Sequence Comparison and Analysis,” MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp 127-149(1988), Alan R. Liss, Inc.

[0117] An example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments. It can also plot a tree showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987); the method is similar to that describedby Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parametersincluding a default gap weight of 3.00, a default gap length weight of0.10, and weighted end gaps.

[0118] Another example of a useful algorithm is the BLAST algorithm,described in Altschul et al., J. Mol. Biol. 215, 403410, (1990) andKarlin et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Methods in Enzymology, 266: 460-480 (1996)]. WU-BLAST-2 usesseveral search parameters, most of which are set to the default values.The adjustable parameters are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSPS2 parameters are dynamic values and are established by the programitself depending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

[0119] An additional useful algorithm is gapped BLAST as reported byAltschul et al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST usesBLOSUM-62 substitution scores; threshold T parameter set to 9; thetwo-hit method to trigger ungapped extensions; charges gap lengths of ka cost of 10+k; X_(u) set to 16, and X_(g) set to 40 for database searchstage and to 67 for the output stage of the algorithms. Gappedalignments are triggered by a score corresponding to ˜22 bits.

[0120] A percent amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

[0121] In a similar manner, “percent (%) nucleic acid sequence identity”with respect to the coding sequence of the polypeptides identifiedherein is defined as the percentage of nucleotide residues in acandidate sequence that are identical with the nucleotide residues inthe coding sequence of the myosin-1F protein. A preferred methodutilizes the BLASTN module of WU-BLAST-2 set to the default parameters,with overlap span and overlap fraction set to 1 and 0.125, respectively.

[0122] The alignment may include the introduction of gaps in thesequences to be aligned. In addition, for sequences which contain eithermore or fewer amino acids than the protein encoded by SEQ ID NO:1, it isunderstood that in one embodiment, the percentage of sequence identitywill be determined based on the number of identical amino acids inrelation to the total number of amino acids. Thus, for example, sequenceidentity of sequences shorter than that shown in the Figure, asdiscussed below, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

[0123] In one embodiment, only identities are scored positively (+1) andall forms of sequence variation including gaps are assigned a value of“0”, which obviates the need for a weighted scale or parameters asdescribed below for sequence similarity calculations. Percent sequenceidentity can be calculated, for example, by dividing the number ofmatching identical residues by the total number of residues of the“shorter” sequence in the aligned region and multiplying by 100. The“longer” sequence is the one having the most actual residues in thealigned region.

[0124] As will be appreciated by those skilled in the art, the sequencesof the present invention may contain sequencing errors. That is, theremay be incorrect nucleosides, frameshifts, unknown nucleosides, or othertypes of sequencing errors in any of the sequences; however, the correctsequences will fall within the homology and stringency definitionsherein.

[0125] In a preferred embodiment, a myosin-1F protein provided hereinhas one or more of the following characteristics: homology to SEQ IDNO:2; the ability to modulate B-lymphocyte activation without modulatingT-lymphocyte activation; the ability to modulate antigen-receptorinduced CD69 expression in B-lymphocytes; the ability to modulateimmunoglobulin heavy chain gene (IgH) promoter activity inB-lymphocytes; the ability to modulate an increase in intracellularcalcium concentration in B-lymphocytes in response to antigen receptorstimulation; the ability to modulate calcineurin activity inB-lymphocytes; and the ability to modulate NFAT activity inB-lymphocytes. Homology and identity to SEQ ID NO:2 can be determined asdescribed above. In one embodiment, homology and identity are determinedby performing a Blastp search in Genbank's non-redundant proteindatabase using default parameters. In another embodiment, homology andidentity are determined using the following database and parameters:Database: Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR; Lambda of 0.316, K of 0.133 andH of 0; Gapped Lambda of 0.27, K of 0.047, and H of 4.94e-324; Matrix isBLOSUM62; Gap Penalties: Existence: 11, Extension: 1.

[0126] In a preferred embodiment, the myosin-1F protein comprises theamino acid sequence set forth in SEQ ID NO:2. The characteristicsdescribed below also apply to other preferred myosin-1F proteinsprovided herein.

[0127] In some preferred embodiments, the myosin-1F protein binds to amyosin-1F binding partner, preferably selected from the group consistingof WASP, BLNK, WASP-IP, Bee1p, Vrp1p, calmodulin, Arp2/3 complex,Acan125, ATP and actin. In a preferred embodiment, the myosin-1F proteinbinds to a myosin-1F binding partner in B-lymphocytes. In anotherpreferred embodiment, the myosin-1F protein binds to a myosin-1F bindingpartner in mast cells. In another preferred embodiment, the myosin-1Fprotein binds to a myosin-1F binding partner in platelets. In anotherpreferred embodiment, the myosin-1F protein binds to a myosin-1F bindingpartner in monocytes. In another preferred embodiment, the myosin-1Fprotein binds to a myosin-1F binding partner in macrophages. In anotherpreferred embodiment, the myosin-1F protein binds to a myosin-1F bindingpartner in peripheral blood lymphocytes. In an especially preferredembodiment, the myosin-1F protein binds to a myosin-1F binding partnerin vitro.

[0128] WASP is the known Wiskott-Aldrich syndrome protein associatedwith Wiskott-Aldrich syndrome (Ramesh et al., Trends Cell Biol.,9:15-19, 1999; Machesky et al., Curr. Biol., 8:1347-1356, 1998).Wiskott-Aldrich syndrome is characterized by a decreased number oflymphocytes and platelets.

[0129] WASP-IP is the known WASP-interacting protein (Ramesh et al.,supra; Ramesh et al., Proc. Nat'l Acad. Sci., 94:14671-14676,1997). WASPand WASP-IP are known to associate with each other to regulate actinassembly (Vetterkind et al., J. Biol. Chem., 30:87-95, 2002).

[0130] Bee1p is a yeast homolog of WASP (Winter et al., Curr. Biol.,9:501-504, 1999) and Vrp1p is a yeast homolog of WASP-IP (Vaduva et al.,J. Biol. Chem., 274:17103-17108, 1999). Beep1p and Vrp1p, as well as theArp2/3 complex, are also involved in actin assembly in yeast (Winter etal., Proc. Natl. Acad. Sci., 96:7288-7293,1999).

[0131] Acan125 is a myosin I binding protein from Acanthamoeba (Xu etal., Proc. Natl. Acad. Sci., 94:3685-3690,1997).

[0132] Calmodulin is a well known calcium-binding protein which binds toand regulates the activity of a large number of diverse proteins(Cheung, Science, 207:19-27, 1980; Hoeflich et al., Cell 108:739-742,2002), including unconventional myosin I proteins.

[0133] ATP is adenosine triphosphate, the known nucleotide containinghydrolyzable phosphodiester bonds. As is known, the hydrolysis of ATP byATPases is coupled to many enzymatic reactions (Stryer, Biochemistry,4^(th) Ed., W. H. Freeman and Co., New York, ISBN 0-7167-2009-4).

[0134] Actin is a well known polar protein which comprises the thinfilaments of the cytoskeleton (Stryer, supra). As is well known in theart, actin polymers are directionally assembled from actin monomers in ahead to tail fashion. The dynamic regulation of actinpolymerization/depolymerization underlies cell motility and shapechanges associated with, among other events, differentiation, division,and transformation.

[0135] BLNK is a well known B-cell linker protein which is involved inthe mediation of B-cell activation. See Fu et al., Immunity, 9:93-103,1998.

[0136] Myosin-1F proteins of the present invention may be shorter orlonger than the amino acid sequence encoded by the nucleic acid sequenceshown in SEQ ID NO:1. Thus, in a preferred embodiment, included withinthe definition of myosin-1F proteins are portions or fragments of theamino acid sequences encoded by the nucleic acid sequences providedherein. In one embodiment herein, fragments of myosin-1F proteins areconsidered myosin-1F proteins if a) they share at least one antigenicepitope; b) have at least the indicated sequence identity; c) andpreferably have myosin-1F protein activity as further defined herein. Insome cases, where the sequence is used diagnostically, that is, when thepresence or absence of myosin-1F nucleic acid is determined, only theindicated sequence identity is required. The nucleic acids of thepresent invention may also be shorter or longer than the sequences inSEQ ID NO:1. The nucleic acid fragments include any portion of thenucleic acids provided herein which have a sequence not exactlypreviously identified; fragments having sequences with the indicatedsequence identity to that portion not previously identified are providedin an embodiment herein.

[0137] In addition, as is more fully outlined below, myosin-1F proteinscan be made that are longer than those depicted in SEQ ID NO:2; forexample, by the addition of epitope or purification tags, the additionof other fusion sequences, or the elucidation of additional coding andnon-coding sequences. As described below, the fusion of a myosin-1Fpeptide to a fluorescent protein, such as Blue Fluorescent Protein (BFP)or Green Fluorescent Protein (GFP), including those of Aquorea andRenilla species, is particularly preferred. In another preferredembodiment, the fluorescent protein is a GFP from Ptilosarcus. Inanother preferred embodiment, the fluorescent protein is a GFP homologuefrom Anthozoa species (Matz et al., Nat. Biotech., 17:969-973, 1999).

[0138] In a preferred embodiment, when a myosin-1F protein is to be usedto generate antibodies, a myosin-1F protein must share at least oneepitope or determinant with the full length protein. By “epitope” or“determinant” herein is meant a portion of a protein which will generateand/or bind an antibody. Thus, in most instances, antibodies made to asmaller myosin-1F protein will be able to bind to the full lengthprotein. In a preferred embodiment, the epitope is unique; that is,antibodies generated to a unique epitope show little or nocross-reactivity. The term “antibody” includes antibody fragments, asare known in the art, including Fab Fab₂, single chain antibodies (Fvfor example), chimeric antibodies, etc., either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies.

[0139] In one embodiment, the antibodies to a myosin-1F protein arecapable of reducing or eliminating the biological function of themyosin-1F proteins described herein, as is described below. That is, theaddition of anti-myosin-1F antibodies (either polyclonal or preferablymonoclonal) to myosin-1F proteins (or cells containing myosin-1Fproteins) may reduce or eliminate their ability to modulate leukocyteand platelet activation. Generally, at least a 25% decrease in activityis preferred, with at least about 50% being particularly preferred andabout a 95-100% decrease being especially preferred. These antibodiesare sometimes referred to herein as function-blocking antibodies.

[0140] In another embodiment, anti-myosin-1F antibodies which increasethe activity of myosin-1F or potentiate the activity of myosin-1F(function activating antibodies) are provided.

[0141] Function-activating and function-blocking antibodies may alterthe ATPase activity of myosin-1F, or alter the affinity of myosin-1F foractin or for another myosin-1F binding partner, or may alter anothermyosin-1F bioactivity.

[0142] The anti-myosin-1F antibodies of the invention bind to myosin-1Fproteins. In a preferred embodiment, the antibodies specifically bind tomyosin-1F proteins. By “specifically bind” herein is meant that theantibodies bind to the protein with a binding constant in the range ofat least 10⁻⁴-10⁻⁶ M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹ M⁻¹.Antibodies are further described below.

[0143] The myosin-1F proteins and myosin-1F nucleic acids of the presentinvention are preferably recombinant. As used herein and further definedbelow, “nucleic acid” may refer to either DNA or RNA, or molecules whichcontain both deoxy- and ribonucleotides. The nucleic acids includegenomic DNA, cDNA and oligonucleotides including sense and anti-sensenucleic acids. Such nucleic acids may also contain modifications in theribose-phosphate backbone to increase stability and half life of suchmolecules in physiological environments.

[0144] The nucleic acid may be double stranded, single stranded, orcontain portions of both double stranded and single stranded sequence.As will be appreciated by those in the art, the depiction of a singlestrand (“Watson”) also defines the sequence of the other strand(“Crick”); thus the sequences depicted in the Figures also include thecomplement of the sequence.

[0145] By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid by endonucleases, in a form not normally found in nature.Thus an isolated myosin-1F nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention. This includes nucleic acids which incorporate into thegenome of a host cell.

[0146] Similarly, a “recombinant protein” is a protein made usingrecombinant techniques, i.e. through the expression of a recombinantnucleic acid as depicted above. A recombinant protein is distinguishedfrom naturally occurring protein by at least one or morecharacteristics. For example, the protein may be isolated or purifiedaway from some or all of the proteins and compounds with which it isnormally associated in its wild type host, and thus may be substantiallypure. For example, an isolated protein is unaccompanied by at least someof the material with which it is normally associated in its naturalstate, preferably constituting at least about 0.5%, more preferably atleast about 5% by weight of the total protein in a given sample. Asubstantially pure protein comprises at least about 75% by weight of thetotal protein, with at least about 80% being preferred, and at leastabout 90% being particularly preferred. The definition includes theproduction of a myosin-1F protein from one organism in a differentorganism or host cell. Alternatively, the protein may be made at asignificantly higher concentration than is normally seen, through theuse of a inducible promoter or high expression promoter. such that theprotein is made at increased concentration levels. Alternatively, theprotein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertions anddeletions, as discussed below.

[0147] In one embodiment, the present invention provides myosin-1Fprotein variants. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a myosin-1F protein, using cassette or PCR mutagenesisor other techniques well known in the art, to produce DNA encoding thevariant, and thereafter expressing the DNA in recombinant cell cultureas outlined above. However, variant myosin-1F protein fragments havingup to about 100-150 residues may be prepared by in vitro synthesis usingestablished techniques. Amino acid sequence variants are characterizedby the predetermined nature of the variation, a feature that sets themapart from naturally occurring allelic or interspecies variation of themyosin-1F protein amino acid sequence. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

[0148] While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed myosin-1F protein variantsscreened for the optimal combination of desired activity. Techniques formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known, for example, M13 primer mutagenesis andPCR mutagenesis. Screening of the mutants is done using assays ofmyosin-1F protein activities.

[0149] Amino acid substitutions are typically of single residues;insertions usually will be on the order of from about 1 to 20 aminoacids, although considerably larger insertions may be tolerated.Deletions range from about 1 to about 20 residues, although in somecases deletions may be much larger.

[0150] Substitutions, deletions, insertions or any combination thereofmay be used to arrive at a final derivative. Generally these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the myosin-1F protein aredesired, substitutions are generally made in accordance with thefollowing chart: CHART I Exemplary Original Residue Substitutions AlaSer Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro HisAsn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile PheMet, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0151] Substantial changes in function or immunological identity aremade by selecting substitutions that are less conservative than thoseshown in Chart I. For example, substitutions may be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example the alpha-helical or beta-sheetstructure; the charge or hydrophomyosin-1Fity of the molecule at thetarget site; or the bulk of the side chain. The substitutions which ingeneral are expected to produce the greatest changes in thepolypeptide's properties are those in which (a) a hydrophilic residue,e.g. seryl or threonyl, is substituted for (or by) a hydrophomyosin-1Fresidue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) acysteine or proline is substituted for (or by) any other residue; (c) aresidue having an electropositive side chain, e.g. lysyl, arginyl, orhistidyl, is substituted for (or by) an electronegative residue, e.g.glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine.

[0152] The variants typically exhibit the same qualitative biologicalactivity and will elicit the same immune response as thenaturally-occurring analogue, although variants also are selected tomodify the characteristics of the myosin-1F proteins as needed. Thevariant may be designed such that the biological activity of themyosin-1F protein is altered. For example, catalytic residues orresidues important for binding to myosin-1F binding partners may bealtered.

[0153] In a preferred embodiment, myosin-1F variant proteins areprovided which lack at least one myosin-1F protein activity. In apreferred embodiment, the myosin-1F variant protein lacks ATPaseactivity. In another preferred embodiment, the myosin-1F variant proteinlacks the ability to bind to at least one myosin-1F binding partnerselected from the group consisting of WASP, BLNK, WASP-IP, Bee1p, Vrp1p,calmodulin, Arp2/3 complex, Acan125, ATP and actin.

[0154] A preferred myosin-1F variant provided herein comprises an SH3domain having a point mutation that diminishes or eliminates one or moremyosin-1F activities, including binding to myosin-1F binding partners.Especially preferred is a myosin-1F variant having a mutation in the SH3domain.

[0155] Another preferred myosin-1F variant provided herein comprises amyosin head domain having a point mutation that diminishes or eliminatesone or more myosin-1F activities, including the ability to hydrolyze ATPand to bind actin in an ATP-dependent manner.

[0156] Another preferred myosin-1F variant provided herein comprises amyosin head domain having a point mutation that diminishes or eliminatesthe ability of myosin-1F to bind ATP.

[0157] A preferred myosin-1F variant provided herein has a pointmutation at a residue which is normally phosphorylated in the context ofmyoain-1F by a protein kinase, preferably a PAK.

[0158] In a preferred embodiment, a myosin-1Fvariant protein providedherein exhibits dominant negative activity, i.e., inhibits the activityof wildtype myosin-1F protein. Such proteins are sometimes referred toherein as dominant negative myosin-1F proteins. Especially preferred arevariant proteins which are capable of inhibiting the ability of wildtypemyosin-1F to modulate lymphocyte activation.

[0159] In a preferred embodiment, the dominant negative myosin-1Fprotein modulates activation of the IgH promoter in lymphocytes.

[0160] In a preferred embodiment, the dominant negative myosin-1Fprotein modulates BCR-induced CD69 expression in B-lymphocytes.

[0161] In a preferred embodiment, the dominant negative myosin-1Fprotein modulates BCR-induced calcium flux in B-lymphocytes.

[0162] Without being bound by theory, in one aspect the dominantnegative myosin-1F protein acts downstream of the BCR.

[0163] In one embodiment, myosin-1F variant proteins are provided whichhave increased activity. Increased activity may be due to a higher levelof catalytic efficiency, a higher affinity for myosin-1F bindingproteins, or a lower level of inhibition, or a combination thereof.

[0164] In a preferred embodiment, such myosin-1F variants modulateB-lymphocyte activation, for example in response to stimuli includingbut not limited to BCR engagement.

[0165] Covalent modifications of myosin-1F polypeptides are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a myosin-1Fpolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N-or C-terminal residues of amyosin-1F polypeptide. Derivatization with bifunctional agents isuseful, for instance, for crosslinking myosin-1F to a water-insolublesupport matrix or surface for use in the method for purifyinganti-myosin-1F antibodies or screening assays, as is more fullydescribed below. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

[0166] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the “-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0167] Another type of covalent modification of the myosin-1Fpolypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencemyosin-1F polypeptide, and/or adding one or more glycosylation sitesthat are not present in the native sequence myosin-1F polypeptide.

[0168] Addition of glycosylation sites to myosin-1F polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence myosin-1Fpolypeptide (for O-linked glycosylation sites). The myosin-1F amino acidsequence may optionally be altered through changes at the DNA level,particularly by mutating the DNA encoding the myosin-1F polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids.

[0169] Another means of increasing the number of carbohydrate moietieson the myosin-1F polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0170] Removal of carbohydrate moieties present on the myosin-1Fpolypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0171] Another type of covalent modification of myosin-1F compriseslinking the myosin-1F polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0172] Myosin-1F polypeptides of the present invention may also bemodified in a way to form chimeric molecules comprising a myosin-1Fpolypeptide fused to another, heterologous polypeptide or amino acidsequence. In one embodiment, such a chimeric molecule comprises a fusionof a myosin-1F polypeptide with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. In apreferred embodiment, such a tag is the “flag tag” described below. Theepitope tag is generally placed at the amino-or carboxyl-terminus of themyosin-1F polypeptide. The presence of such epitope-tagged forms of amyosin-1F polypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the myosin-1Fpolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a myosin-1F polypeptide with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule as discussed further below.

[0173] Various tag polypeptides and their respective antibodies are wellknown in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0174] In an embodiment herein, myosin-1F protein family members andmyosin-1F proteins from other organisms are cloned and expressed asoutlined below. Thus, probe or degenerate polymerase chain reaction(PCR) primer sequences may be used to find other related myosin-1Fproteins from humans or other organisms. As will be appreciated by thosein the art, particularly useful probe and/or PCR primer sequencesinclude the unique areas of the myosin-1F nucleic acid sequence. As isgenerally known in the art, preferred PCR primers are from about 15 toabout 35 nucleotides in length, with from about 20 to about 30 beingpreferred, and may contain inosine as needed. The conditions for the PCRreaction are well known in the art. It is therefore also understood thatprovided along with the sequences in the sequences listed herein areportions of those sequences, wherein unique portions of 15 nucleotidesor more are particularly preferred. The skilled artisan can routinelysynthesize or cut a nucleotide sequence to the desired length.

[0175] Once isolated from its natural source, e.g., contained within aplasmid or other vector or excised therefrom as a linear nucleic acidsegment, the recombinant myosin-1F nucleic acid can be further-used as aprobe to identify and isolate other myosin-1F nucleic acids. It can alsobe used as a “precursor” nucleic acid to make modified or variantmyosin-1F nucleic acids and proteins.

[0176] Of course, as will be recognized by the artisan, PCR may also beused to obtain large quantities of a desired myosin-1F nucleic acid froma source comprising such a myosin-1F nucleic acid.

[0177] Using the nucleic acids of the present invention which encode amyosin-1F protein, a variety of expression vectors are made. Theexpression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a host genome. Generally, theseexpression vectors include transcriptional and translational regulatorynucleic acid operably linked to the nucleic acid encoding the myosin-1Fprotein. The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0178] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. As anotherexample, operably linked refers to DNA sequences linked so as to becontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice. The transcriptionaland translational regulatory nucleic acid will generally be appropriateto the host cell used to express the myosin-1F protein; for example,transcriptional and translational regulatory nucleic acid sequences fromBacillus are preferably used to express the myosin-1F protein inBacillus. Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of host cells.

[0179] In general, the transcriptional and translational regulatorysequences may include, but are not limited to, promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, and enhancer or activatorsequences. In a preferred embodiment, the regulatory sequences include apromoter and transcriptional start and stop sequences.

[0180] Promoter sequences encode either constitutive or induciblepromoters. The promoters may be either naturally occurring promoters orhybrid promoters. Hybrid promoters, which combine elements of more thanone promoter, are known in the art, and are useful in the presentinvention.

[0181] In addition, the expression vector may comprise additionalelements. For example, the expression vector may have two replicationsystems, thus allowing it to be maintained in two organisms, for examplein mammalian or insect cells for expression and in a prokaryotic hostfor cloning and amplification. Furthermore, for integrating expressionvectors, the expression vector contains at least one sequence homologousto the host cell genome, and preferably two homologous sequences whichflank the expression construct. The integrating vector may be directedto a specific locus in the host cell by selecting the appropriatehomologous sequence for inclusion in the vector. Constructs forintegrating vectors are well known in the art.

[0182] In addition, in a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selection genes are well known in the art and will vary withthe host cell used.

[0183] A preferred expression vector system is a retroviral vectorsystem such as is generally described in PCT/US97/01019 andPCT/US97/01048, both of which are hereby expressly incorporated byreference.

[0184] Myosin-1F proteins of the present invention are produced byculturing a host cell transformed with an expression vector containingmyosin-1F nucleic acid encoding a myosin-1F protein, under theappropriate conditions to induce or cause expression of the myosin-1Fprotein. The conditions appropriate for myosin-1F protein expressionwill vary with the choice of the expression vector and the host cell,and will be easily ascertained by one skilled in the art through routineexperimentation. For example, the use of constitutive promoters in theexpression vector will require optimizing the growth and proliferationof the host cell, while the use of an inducible promoter requires theappropriate growth conditions for induction. In addition, in someembodiments, the timing of the harvest is important. For example, thebaculoviral systems used in insect cell expression are lytic viruses,and thus harvest time selection can be crucial for product yield.

[0185] Appropriate host cells include yeast, bacteria, archebacteria,fungi, and insect and animal cells, including mammalian cells. Ofparticular interest are Drosophila melangaster cells, Saccharomycescerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells,fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid andlymphoid cell lines.

[0186] In a preferred embodiment, the myosin-1F proteins are expressedin mammalian cells. Mammalian expression systems are also known in theart, and include retroviral systems. A mammalian promoter is any DNAsequence capable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for myosin-1F proteininto mRNA. A promoter will have a transcription initiating region, whichis usually placed proximal to the 5′ end of the coding sequence, andusually a TATA box, typically located 25-30 base pairs upstream of thetranscription initiation site. The TATA box is thought to direct RNApolymerase 11 to begin RNA synthesis at the correct site. However,TATA-free transcription initiation is also well known. A mammalianpromoter will also contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box if present. An upstream promoter element determines the rate atwhich transcription is initiated and can act in either orientation. Ofparticular use as mammalian promoters are the promoters from mammalianviral genes, since the viral genes are often highly expressed and have abroad host range. Examples include the SV40 early promoter, mousemammary tumor virus LTR promoter, adenovirus major late promoter, herpessimplex virus promoter, and the CMV promoter.

[0187] Typically, transcription termination and polyadenylationsequences recognized by mammalian cells are regulatory regions located3′ to the translation stop codon and thus, together with the promoterelements, flank the coding sequence. The 3′ terminus of the mature mRNAis formed by site-specific post-transcriptional cleavage andpolyadenylation. Examples of transcription terminator and polyadenlytionsignals include those derived form SV40.

[0188] The methods of introducing exogenous nucleic acid into mammalianhosts, as well as other hosts, are well known in the art, and will varywith the host cell used. Techniques include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, viral infection,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei.

[0189] In a preferred embodiment, myosin-1F proteins are expressed inbacterial systems. Bacterial expression systems are well known in theart.

[0190] A suitable bacterial promoter is any nucleic acid sequencecapable of binding bacterial RNA polymerase and initiating thedownstream (3′) transcription of the coding sequence of myosin-1Fprotein into mRNA. A bacterial promoter has a transcription initiationregion which is usually placed proximal to the 5′ end of the codingsequence. This transcription initiation region typically includes an RNApolymerase binding site and a transcription initiation site. Sequencesencoding metabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

[0191] In addition to a functioning promoter sequence, an efficientribosome binding site is desirable. In E. coli, the ribosome bindingsite is called the Shine-Delgarno (SD) sequence and includes aninitiation codon and a sequence 3-9 nucleotides in length located 3-11nucleotides upstream of the initiation codon.

[0192] The expression vector may also include a signal peptide sequencethat provides for secretion of the myosin-1F protein in bacteria. Thesignal sequence typically encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell, as is well known in the art. The protein is either secretedinto the growth media (gram-positive bacteria) or into the periplasmicspace, located between the inner and outer membrane of the cell(gram-negative bacteria).

[0193] The bacterial expression vector may also include a selectablemarker gene to allow for the selection of bacterial strains that havebeen transformed. Suitable selection genes include genes which renderthe bacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

[0194] These components are assembled into expression vectors.Expression vectors for bacteria are well known in the art, and includevectors for Bacillus subtilis, E. coli, Streptococcus cremoris, andStreptococcus lividans, among others.

[0195] The bacterial expression vectors are transformed into bacterialhost cells using techniques well known in the art, such as calciumchloride treatment, electroporation, and others.

[0196] In one embodiment, myosin-1F proteins are produced in insectcells. Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

[0197] In a preferred embodiment, myosin-1F protein is produced in yeastcells. Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred promoter sequences for expression inyeast include the inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

[0198] The myosin-1F protein may also be made as a fusion protein, usingtechniques well known in the art. Thus, for example, for the creation ofmonoclonal antibodies, if the desired epitope is small, the myosin-1Fprotein may be fused to a carrier protein to form an immunogen.Alternatively, the myosin-1F protein may be made as a fusion protein toincrease expression, or for other reasons. For example, when themyosin-1F protein is a myosin-1F peptide, the nucleic acid encoding thepeptide may be linked to other nucleic acid for expression purposes.Similarly, myosin-1F proteins of the invention can be linked to proteinlabels, such as green fluorescent protein (GFP), red fluorescent protein(RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP),etc.

[0199] In one embodiment, the myosin-1F nucleic acids, proteins andantibodies of the invention are labeled. By “labeled” herein is meantthat a compound has at least one element, isotope or chemical compoundattached to enable the detection of the compound. In general, labelsfall into three classes: a) isotopic labels, which may be radioactive orheavy isotopes; b) immune labels, which may be antibodies or antigens;and c) colored or fluorescent dyes. The labels may be incorporated intothe compound at any position.

[0200] In a preferred embodiment, the myosin-1F protein is purified orisolated after expression. Myosin-1F proteins may be isolated orpurified in a variety of ways known to those skilled in the artdepending on what other components are present in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the myosin-1F protein may be purified using a standardanti-myosin-1F antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.For general guidance in suitable purification techniques, see Scopes,R., Protein Purification, Springer-Verlag, NY (1982). The degree ofpurification necessary will vary depending on the use of the myosin-1Fprotein. In some instances no purification will be necessary.

[0201] Once expressed and purified if necessary, the myosin-1F proteinsand nucleic acids are useful in a number of applications.

[0202] The nucleotide sequences (or their complement) encoding myosin-1Fproteins have various applications in the art of molecular biology,including uses as hybridization probes, in chromosome and gene mappingand in the generation of anti-sense RNA and DNA. Myosin-1F proteinnucleic acid will also be useful for the preparation of myosin-1Fproteins by the recombinant techniques described herein.

[0203] The full-length native sequence myosin-1F protein gene, orportions thereof, may be used as hybridization probes for a cDNA libraryor genomic DNA library to isolate other genes (for instance, thoseencoding naturally-occurring variants of myosin-1F protein or myosin-1Fprotein from other species) which have a desired sequence identity tothe myosin-1F protein coding sequence. Optionally, the length of theprobes will be about 20 to about 50 bases. The hybridization probes maybe derived from the nucleotide sequences herein or from genomicsequences including promoters, enhancer elements and introns of nativesequences as provided herein. By way of example, a screening method willcomprise isolating the coding region of the myosin-1F protein gene usingthe known DNA sequence to synthesize a selected probe of about 40 bases.Hybridization probes may be labeled by a variety of labels, includingradionucleotides such as ³²P or ³⁵S, or enzymatic labels such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems. Labeled probes having a sequence complementary to that of themyosin-1F protein gene of the present invention can be used to screenlibraries of human cDNA, genomic DNA or mRNA to determine which membersof such libraries the probe hybridizes.

[0204] Nucleotide sequences encoding a myosin-1F protein can also beused to construct hybridization probes for mapping the gene whichencodes that myosin-1F protein and for the genetic analysis ofindividuals with genetic disorders. The nucleotide sequences providedherein may be mapped to a chromosome and specific regions of achromosome using known techniques, such as in situ hybridization,linkage analysis against known chromosomal markers, and hybridizationscreening with libraries.

[0205] Nucleic acids which encode myosin-1F protein or its modifiedforms can also be used to generate either transgenic animals or “knockout” animals which, in turn, are useful in the development and screeningof therapeutically useful reagents. A transgenic animal (e.g., a mouseor rat) is an animal having cells that contain a transgene, whichtransgene was introduced into the animal or an ancestor of the animal ata prenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding a myosin-1F protein can beused to clone genomic DNA encoding a myosin-1F protein in accordancewith established techniques and the genomic sequences used to generatetransgenic animals that contain cells which express the desiredmyosin-1F DNA. In another embodiment, cDNA is used in the formation of atransgene. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for the myosin-1F proteintransgene expression with tissue-specific enhancers. Transgenic animalsthat include a copy of a transgene encoding a myosin-1F proteinintroduced into the germ line of the animal at an embryonic stage can beused to examine the effect of increased expression of the desirednucleic acid. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this facet of theinvention, an animal is treated with the reagent and a reduced incidenceof the pathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

[0206] Alternatively, non-human homologues of the myosin-1F protein canbe used to construct a myosin-1F protein “knock out” animal which has adefective or altered gene encoding a myosin-1F protein as a result ofhomologous recombination between the endogenous gene encoding amyosin-1F protein and altered genomic DNA encoding a myosin-1F proteinintroduced into an embryonic cell of the animal. For example, cDNAencoding a myosin-1F protein can be used to clone genomic DNA encoding amyosin-1F protein in accordance with established techniques. A portionof the genomic DNA encoding a myosin-1F protein can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of themyosin-1F protein.

[0207] It is understood that the models described herein can be varied.For example, “knock-in” models can be formed, or the models can becell-based rather than animal models.

[0208] Nucleic acid encoding the myosin-1F polypeptides, antagonists oragonists may also be used in gene therapy. In gene therapy applications,genes are introduced into cells in order to achieve in vivo synthesis ofa therapeutically effective genetic product, for example for replacementof a defective gene. “Gene therapy” includes both conventional genetherapy where a lasting effect is achieved by a single treatment, andthe administration of gene therapeutic agents, which involves the onetime or repeated administration of a therapeutically effective DNA ormRNA. Antisense RNAs and DNAs can be used as therapeutic agents forblocking the expression of certain genes in vivo. It has already beenshown that short antisense oligonucleotides can be imported into cellswhere they act as inhibitors, despite their low intracellularconcentrations caused by their restricted uptake by the cell membrane.(Zamecnik et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 [1986]). Theoligonucleotides can be modified to enhance their uptake, e.g. bysubstituting their negatively charged phosphodiester groups by unchargedgroups.

[0209] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al., Trends inBiotechnology 1,205-210 [1993]). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

[0210] In a preferred embodiment, the myosin-1F proteins, nucleic acids,variants, modified proteins, cells and/or transgenics containing thesaid nucleic acids or proteins are used in screening assays.Identification of the myosin-1F proteins provided herein permits thedesign of drug screening assays for compounds that bind myosin-1Fproteins, interfere with myosin-1F protein binding, modulate myosin-1Factivity, and modulate B-lymphocyte activation.

[0211] The assays described herein preferably utilize human myosin-1Fprotein, although other mammalian proteins may also be used, includingrodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows,sheep, pigs, horses, etc.) and primates. These latter embodiments may bepreferred in the development of animal models of human disease. In someembodiments, as outlined herein, truncated myosin-1F proteins may beused.

[0212] In a preferred embodiment, the methods comprise combining amyosin-1F protein and a candidate bioactive agent, and determining thebinding of the candidate agent to the myosin-1F protein. In otherembodiments, further discussed below, binding interference orbioactivity is determined.

[0213] The term “candidate bioactive agent” or “exogeneous compound” asused herein describes any molecule, e.g., protein, small organicmolecule, carbohydrates (including polysaccharides), polynucleotide,lipids, etc. Generally a plurality of assay mixtures are run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection. In addition, positive controls, i.e. the use ofagents known to bind myosin-1F protein, may be used.

[0214] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons, more preferably between 100 and 2000, more preferably betweenabout 100 and about 1250, more preferably between about 100 and about1000, more preferably between about 100 and about 750, more preferablybetween about 200 and about 500 daltons. Candidate agents comprisefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides.

[0215] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification,amidification to produce structural analogs.

[0216] In a preferred embodiment, a library of different candidatebioactive agents are used. Preferably, the library should provide asufficiently structurally diverse population of randomized agents toeffect a probabilistically sufficient range of diversity to allowbinding to a particular target. Accordingly, an interaction libraryshould be large enough so that at least one of its members will have astructure that gives it affinity for the target. Although it isdifficult to gauge the required absolute size of an interaction library,nature provides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. A library of all combinations of a peptide 7 to 20 aminoacids in length, such as generally proposed herein, has the potential tocode for 20⁷ (10⁹) to 20⁰. Thus, with libraries of 10⁷ to 10⁹ differentmolecules the present methods allow a “working” subset of atheoretically complete interaction library for 7 amino acids, and asubset of shapes for the 20²⁰ library. Thus, in a preferred embodiment,at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ andmost preferably at least 10⁹ different sequences are simultaneouslyanalyzed in the subject methods. Preferred methods maximize library sizeand diversity.

[0217] In a preferred embodiment, the candidate bioactive agents areproteins. By “protein” herein is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Chemical blocking groups orother chemical substituents may also be added.

[0218] In a preferred embodiment, the candidate bioactive agents arenaturally occurring proteins or fragments of naturally occurringproteins. Thus, for example, cellular extracts containing proteins, orrandom or directed digests of proteinaceous cellular extracts, may beused. In this way libraries of procaryotic and eukaryotic proteins maybe made for screening in the systems described herein. Particularlypreferred in this embodiment are libraries of bacterial, fungal, viral,and mammalian proteins, with the latter being preferred, and humanproteins being especially preferred.

[0219] In a preferred embodiment, the candidate bioactive agents arepeptides of from about 5 to about 30 amino acids, with from about 5 toabout 20 amino acids being preferred, and from about 7 to about 15 beingparticularly preferred, and 12 and 18 amino acids being most preferred.The peptides may be digests of naturally occurring proteins as isoutlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

[0220] In one embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

[0221] In a preferred embodiment, the candidate bioactive agents arenucleic acids. By “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein means at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805(1984), Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); andPauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989)), O-methylphophoroamiditelinkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc., 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al.,Nature, 380:207 (1996), all of which are incorporated by reference)).Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995)); non-ionicbackbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141;and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English,30:423 (1991); Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988);Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, etal., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclicsugars, as well as “locked nucleic acids”, are also included within thedefinition of nucleic acids (see Jenkins, et al., Chem. Soc. Rev.,(1995) pp. 169-176). Several nucleic acid analogs are described inRawls, C & E News, Jun. 2, 1997, page 35. All of these references arehereby expressly incorporated by reference. These modifications of theribose-phosphate backbone may be done to facilitate the addition ofadditional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments. In addition,mixtures of naturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. Thenucleic acids may be single stranded or double stranded, as specified,or contain portions of both double stranded or single stranded sequence.The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,where the nucleic acid contains any combination of deoxyribo- andribo-nucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine,isocytosine, isoguanine, etc.

[0222] For an example of how nucleic acids may be used as candidateagents to screen for a change in phenotype, see Holland et al., J. Exp.Med., 194:1263-1276, 2001. See also Hitoshi et al., Immunity, 8:461471,1998. Both of which are expressly incorporated herein by reference.

[0223] As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryotic genomes may be used as is outlined above forproteins.

[0224] In a preferred embodiment, the candidate bioactive agents areorganic chemical moieties or small molecule chemical compositions, awide variety of which are available in the literature.

[0225] In a preferred embodiment, the candidate bioactive agents arelinked to a fusion partner. By “fusion partner” or “functional group”herein is meant a sequence that is associated with the candidatebioactive agent, that confers upon all members of the library in thatclass a common function or ability. Fusion partners can be heterologous(i.e. not native to the host cell), or synthetic (not native to anycell). Suitable fusion partners include, but are not limited to: a)presentation structures, which provide the candidate bioactive agents ina conformationally restricted or stable form; b) targeting sequences,which allow the localization of the candidate bioactive agent into asubcellular or extracellular compartment; c) rescue sequences whichallow the purification or isolation of either the candidate bioactiveagents or the nucleic acids encoding them; d) stability sequences, whichconfer stability or protection from degradation to the candidatebioactive agent or the nucleic acid encoding it, for example resistanceto proteolytic degradation; e) dimerization sequences, to allow forpeptide dimerization; or f) any combination of a), b), c), d), and e),as well as linker sequences as needed.

[0226] Generally, in a preferred embodiment of the methods herein, forexample for binding assays, the myosin-1F protein or the candidate agentis non-diffusibly bound to an insoluble support having isolated samplereceiving areas (e.g. a microtiter plate, an array, etc.). The insolublesupports may be made of any composition to which the compositions can bebound, is readily separated from soluble material, and is otherwisecompatible with the overall method of screening. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports include microtiter plates, arrays, membranesand beads. These are typically made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon or nitrocellulose, teflon™, etc.Microtiter plates and arrays are especially convenient because a largenumber of assays can be carried out simultaneously, using small amountsof reagents and samples. In some cases magnetic beads and the like areincluded. The particular manner of binding of the composition is notcrucial so long as it is compatible with the reagents and overallmethods of the invention, maintains the activity of the composition andis nondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the protein or agent on the surface, etc. Following bindingof the protein or agent, excess unbound material is removed by washing.The sample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety. Also included in this invention are screening assays whereinsolid supports are not used; examples of such are described below.

[0227] In a preferred embodiment, the myosin-1F protein is bound to thesupport, and a candidate bioactive agent is added to the assay.Alternatively, the candidate agent is bound to the support and themyosin-1F protein is added. Novel binding agents include specificantibodies, non-natural binding agents identified in screens of chemicallibraries, peptide analogs, etc. Of particular interest are screeningassays for agents that have a low toxicity for human cells. A widevariety of assays may be used for this purpose, including labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, functional assays(phosphorylation assays, etc.) and the like.

[0228] The determination of the binding of the candidate bioactive agentto the myosin-1F protein may be done in a number of ways. In a preferredembodiment, the candidate bioactive agent is labelled, and bindingdetermined directly. For example, this may be done by attaching all or aportion of the myosin-1F protein to a solid support, adding a labelledcandidate agent (for example a fluorescently labeled agent), washing offexcess reagent, and determining whether the label is present on thesolid support. Various blocking and washing steps may be utilized as isknown in the art.

[0229] By “labeled” herein is meant that the compound is either directlyor indirectly labeled with a label which provides a detectable signal,e.g. radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined herein. The label can directly or indirectlyprovide a detectable signal.

[0230] In some embodiments, only one of the components is labeled. Forexample, the proteins (or proteinaceous candidate agents) may be labeledat tyrosine positions using ¹²⁵I, or with fluorophores. Alternatively,more than one component may be labeled with different labels; using ¹²⁵Ifor the proteins, for example, and a fluorophor for the candidateagents.

[0231] In a preferred embodiment, the binding of the candidate bioactiveagent is determined through the use of competitive binding assays. Inthis embodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. myosin-1F protein), such as an antibody. In apreferred embodiment, the competitor is selected from the groupconsisting of WASP, BLNK, WASP-IP, Bee1p, Vrp1p, calmodulin, Arp2/3complex, Acan125, ATP and actin. Under certain circumstances, there maybe competitive binding as between the bioactive agent and the bindingmoiety, with the binding moiety displacing the bioactive agent. Thisassay can be used to determine candidate agents which interfere withbinding of myosin-1F to its binding partner. “Binding interference”, orgrammatical equivalents, as used herein means that native binding of themyosin-1F protein differs in the presence of the candidate agent. Thebinding can be eliminated or can be with a reduced affinity. In oneembodiment, interference is caused by, for example, a conformationalchange, rather than direct competition for the native binding site.

[0232] In one embodiment, the candidate bioactive agent is labeled.Either the candidate bioactive agent, or the competitor, or both, isadded first to the protein for a time sufficient to allow binding, ifpresent. Incubations may be performed at any temperature whichfacilitates optimal activity, typically between 4° C. and 40° C.Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high through put screening. Typicallybetween 0.1 and 1 hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

[0233] In a preferred embodiment, the competitor is added first,followed by the candidate bioactive agent. Displacement of thecompetitor is an indication that the candidate bioactive agent isbinding to the myosin-1F protein and thus is capable of binding to, andpotentially modulating, the activity of the myosin-1F protein. In thisembodiment, either component can be labeled. Thus, for example, if thecompetitor is labeled, the presence of label in the wash solutionindicates displacement by the agent. Alternatively, if the candidatebioactive agent is labeled, the presence of the label on the supportindicates displacement.

[0234] In an alternative embodiment, the candidate bioactive agent isadded first, with incubation and washing, followed by the competitor.The absence of binding by the competitor may indicate that the bioactiveagent is bound to the myosin-1F protein with a higher affinity. Thus, ifthe candidate bioactive agent is labeled, the presence of the label onthe support, coupled with a lack of competitor binding, may indicatethat the candidate agent is capable of binding to the myosin-1F protein.

[0235] In a preferred embodiment, the methods comprise differentialscreening to identity bioactive agents that are capable of modulatingthe activity of the myosin-1F proteins. In one embodiment, the methodscomprise combining a myosin-1F protein and a competitor in a firstsample. A second sample comprises a candidate bioactive agent, amyosin-1F protein and a competitor. The binding of the competitor isdetermined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to the myosin-1F protein and modulating its activity. That is,if the binding of the competitor is different in the second samplerelative to the first sample, the agent is capable of binding to themyosin-1F protein and modulating its activity.

[0236] Alternatively, a preferred embodiment utilizes differentialscreening to identify drug candidates that bind to the native myosin-1Fprotein, but cannot bind to modified myosin-1F proteins. The structureof the myosin-1F protein may be modeled, and used in rational drugdesign to synthesize agents that interact with a catalytic, binding, orregulatory domain.

[0237] Positive controls and negative controls may be used in theassays. Preferably all control and test samples are performed in atleast triplicate to obtain statistically significant results. Incubationof all samples is for a time sufficient for the binding of the agent tothe protein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

[0238] A variety of other reagents may be included in the screeningassays. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc which may be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Also reagents that otherwise improve the efficiency of theassay, such as protease inhibitors, nuclease inhibitors, anti-microbialagents, etc., may be used. The mixture of components may be added in anyorder that provides for the requisite binding.

[0239] Screening for agents that modulate the activity of a myosin-1Fprotein may also be done. In a preferred embodiment, methods forscreening for a bioactive agent capable of modulating the activity of amyosin-1F protein comprise the steps of adding a candidate bioactiveagent to a sample of a myosin-1F protein and determining an alterationin the biological activity of the myosin-1F protein. “Modulating theactivity of a myosin-1F protein” includes an increase in activity, adecrease in activity, or a change in the type or kind of activitypresent. Thus, in this embodiment, the candidate agent may bind to amyosin-1F protein (although this may not be necessary), and should alterits biological or biochemical activity as defined herein. The methodsinclude in vivo screening of cells for alterations in the presence,cellular distribution, subcellular distribution, activity or amount ofmyosin-1F protein.

[0240] By “myosin-1F protein activity” or grammatical equivalents hereinis meant at least one of the myosin-1F protein's biological activities,including, but not limited to, modulation of B-lymphocyte activation;modulation of lymphocyte activation by antigen; modulation of B-celldifferentiation; modulation of B-lymphocyte proliferation; modulation ofIgM and IgG induction in B-lymphocytes; modulation of calcium fluxinduced by BCR stimulation; modulation of immunoglobulin heavy chaingene promoter activity in lymphocytes; modulation of NFAT activity inB-lymphocytes; modulation of immunoglobulin secretion by B-lymphocytes;modulation of cytokine production in B-lymphocytes; modulation ofsurface protein expression including CD69, CD23, CD80 and CD86 inB-lymphocytes; modulation of intracellular calcium concentration inB-lymphocytes; modulation of intracellular calcium increase in responseto antigen-receptor activation in B-lymphocytes; modulation ofcalcineurin activity in B-lymphocytes; modulation of calcineurinactivity induction by antigen receptor activation in B-lymphocytes;binding to calmodulin, actin, BLNK, ATP, Arp2/3, Bee1p, WASP, WASP-IP,or Vrp1p; ATPase activity; ATP-dependent actin binding; ATP-independentactin binding; actin polymerization-inducing activity; and ability tomodulate B-lymphocyte activation without modulating T-lymphocyteactivation.

[0241] In a preferred embodiment, the activity of the myosin-1F proteinis decreased; in another preferred embodiment, the activity of themyosin-1F protein is increased. Thus, bioactive agents that areantagonists are preferred in some embodiments, and bioactive agents thatare agonists are preferred in other embodiments.

[0242] In an especially preferred embodiment, methods of screening forcandidate bioactive agents capable of modulating the ATPase activity ofa myosin-1F protein are provided. In a preferred embodiment, the methodsinvolve incubating myosin-1F or a sample comprising myosin-1F withγ³²P-labelled ATP and separating ³²P labelled inorganic phosphate fromunreacted γ³²P-labelled ATP by thin layer chromatography to determinethe extent of ATP hydrolysis. Such methods are well known, for example,see Burlacu et al., Biophys. J., 72:263-271, 1997. See also Bikle etal., J. Biol. Chem., 271:9075-9083, 1996.

[0243] In another especially preferred embodiment, methods of screeningfor candidate bioactive agents capable of modulating thecalmodulin-binding activity of a myosin-1F protein are provided. Assaysfor determining calmodulin binding are known in the art, for example,see Coluccio, J. Cell Sci., 107:2279-2284, 1994.

[0244] In another especially preferred embodiment, methods of screeningfor candidate bioactive agents capable of modulating the actin-bindingactivity of a myosin-1F protein are provided. Methods for determiningactin-myosin interactions are well known in the art, for example, seeBikle et al., J. Biol. Chem., 271:9075-9083, 1996.

[0245] In another preferred embodiment, methods of screening forcandidate bioactive agents capable of modulating the actinpolymerization-inducing activity of a myosin-1F protein are provided. Ina preferred embodiment, the methods involve performing actin assemblyassays in the presence of myosin-1F protein. Such assays employing yeastcells are known, for example, see Lechler et al., J. Cell Biol.,148:363-373, 2000. Visual in vitro assays for actin polymerization arealso known, for example, see Geli et al., EMBO J., 19:42814291, 2000.

[0246] Methods for screening for agents that modulate B-cell activationare also provided herein.

[0247] In a preferred embodiment, the methods comprise determining theability of a candidate agent to bind to myosin-1F.

[0248] In a preferred embodiment, the methods comprise detecting bindingof candidate agent to myosin-1F protein, contacting the candidatebioactive agent to a B-lymphocyte, and determining lymphocyte activationin the presence of said candidate agent. In a preferred embodiment,lymphocyte activation in the presence and absence of candidate agent isdetermined. In a preferred embodiment, an agent that normally induceslymphocyte activation is used. Lymphocyte activation is measured in thepresence, and optionally, the absence of candidate agent followingcontact with the agent that normally induces lymphocyte activation. Apreferred activation agent for use with B lymphocytes is anti-IgMantibody.

[0249] It will be understood that while agents that normally inducelymphocyte activation (i.e., activation agents) are used, the screeningmethod is designed to identify agents that are capable of inhibitinglymphocyte activation. Accordingly, the presence of a bioactive agentthat is capable of inhibiting lymphocyte activation may precludeactivation of the lymphocyte by the activation agent. Such agents arenevertheless referred to herein as activation agents, and the step ofcontacting the cells with such an activation agent is frequentlyreferred to herein as “inducing lymphocyte activation”, even though acandidate bioactive agent may inhibit such activation by the agent. Thisnomenclature applies to the methods that follow as well.

[0250] In a preferred embodiment, the myosin-1F protein used in themethod comprises a tail domain, including an SH3 domain, but lacks amyosin head domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 718-1098 in SEQ ID NO:2. An especiallypreferred myosin-1F protein for use in the method consists essentiallyof amino acids 718-1098 in SEQ ID NO:2.

[0251] In another preferred embodiment, the myosin-1F protein used inthe method comprises an IQ domain and a tail domain, including an SH3domain, but lacks a myosin head domain. A preferred myosin-1F proteinfor use in the method consists essentially of an amino acid sequencehaving at least about 90%, more preferably at least about 95%, morepreferably at least about 98% identity to residues 695-1098 in SEQ IDNO:2. An especially preferred myosin-1F protein for use in the methodconsists essentially of amino acids 695-1098 in SEQ ID NO:2.

[0252] In another preferred embodiment, the myosin-1F protein used inthe method comprises an IQ domain and a tail domain, including an SH3domain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to residues 617-1098 in SEQ ID NO:2. An especially preferredmyosin-1F protein for use in the method consists essentially of aminoacids 617-1098 in SEQ ID NO:2.

[0253] In another preferred embodiment, the myosin-1F protein used inthe method comprises a TH2 and SH3 domain, but lacks an IQ domain and amyosin head domain.

[0254] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain and an IQ domain, but lacks atail domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 1-717, 12-717 or 19-717 in SEQ ID NO:2. Anespecially preferred myosin-1F protein for use in the method consistsessentially of amino acids 1-717, 12-717 or 19-717 in SEQ ID NO:2.

[0255] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, but lacks an IQ domain and atail domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 19-677, 1-677, 12-691, or 1-691 in SEQ ID NO:2.An especially preferred myosin-1F protein for use in the method consistsessentially of amino acids 19-677, 1-677, 12-691, or 1-691 in SEQ IDNO:2.

[0256] In another preferred embodiment, the myosin-1F protein usedconsists essentially of a partial myosin head domain. In a preferredembodiment, such a myosin-1F protein consists essentially of an aminoacid sequence having at least about 85%, more preferably at least about90%, more preferably at least about 95%, more preferably at least about98% identity to the amino acid sequence set forth by residues 260-677 or260-691 in SEQ ID NO:2. In an especially preferred embodiment, such amyosin-1F protein consists essentially of the amino acid sequence setforth by residues 260-677 or 260-691 in SEQ ID NO:2.

[0257] In another preferred embodiment, the myosin-1F protein consistsessentially of a partial myosin head domain, an IQ domain, and a taildomain. In a preferred embodiment, such a myosin-1F protein consistsessentially of an amino acid sequence having at least about 85%, morepreferably at least about 90%, more preferably at least about 95%, morepreferably at least about 98% identity to the amino acid sequence setforth by residues 260-1098 in SEQ ID NO:2. In an especially preferredembodiment, such a myosin-1F protein consists essentially of the aminoacid sequence set forth by residues 260-1098 in SEQ ID NO:2. In anotherespecially preferred embodiment, such a myosin-1F protein consistsessentially of the amino acid sequence set forth by SEQ ID NO:4.

[0258] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, an IQ domain, and a taildomain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to SEQ ID NO:2. An especially preferred myosin-1F protein foruse in the method comprises SEQ ID NO:2.

[0259] Bioactive agents that inhibit B-lymphocyte activation in theseassays are useful as immunosuppressants.

[0260] By immunosuppressant is meant an agent that suppresses the body'sability to react to an antigen.

[0261] In another preferred embodiment, the methods comprise determiningthe ability of a candidate agent to modulate the binding of myosin-1F toa myosin-1F binding partner.

[0262] In a preferred embodiment, the methods comprise detectingmodulation of the binding of myosin-1F protein to a myosin-1F bindingpartner in the presence of candidate agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining lymphocyte activationin the presence of said candidate agent. In a preferred embodiment,lymphocyte activation in the presence and absence of candidate agent isdetermined. In a preferred embodiment, an agent that normally induceslymphocyte activation is used. Lymphocyte activation is measured in thepresence, and optionally, the absence of candidate agent followingcontact with the agent that normally induces lymphocyte activation. Apreferred activation agent for use with B lymphocytes is anti-IgMantibody.

[0263] In a preferred embodiment, the myosin-1F binding partner used isselected from the group consisting of WASP, BLNK, WASP-IP, Bee1p, Vrp1p,calmodulin, Arp2/3 complex, Acan125, ATP and actin.

[0264] In a preferred embodiment, the myosin-1F protein used in themethod comprises a tail domain, including an SH3 domain, but lacks amyosin head domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 718-1098 in SEQ ID NO:2. An especiallypreferred myosin-1F protein for use in the method consists essentiallyof amino acids 718-1098 in SEQ ID NO:2.

[0265] In another preferred embodiment, the myosin-1F protein used inthe method comprises an IQ domain and a tail domain, including an SH3domain, but lacks a myosin head domain. A preferred myosin-1F proteinfor use in the method consists essentially of an amino acid sequencehaving at least about 90%, more preferably at least about 95%, morepreferably at least about 98% identity to residues 695-1098 in SEQ IDNO:2. An especially preferred myosin-1F protein for use in the methodconsists essentially of amino acids 695-1098 in SEQ ID NO:2.

[0266] In another preferred embodiment, the myosin-1F protein used inthe method comprises an IQ domain and a tail domain, including an SH3domain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to residues 617-1098 in SEQ ID NO:2. An especially preferredmyosin-1F protein for use in the method consists essentially of aminoacids 617-1098 in SEQ ID NO:2.

[0267] In another preferred embodiment, the myosin-1F protein used inthe method comprises a TH2 and TH3 domain, but lacks an IQ domain and amyosin head domain.

[0268] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain and an IQ domain, but lacks atail domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 1-717, 12-717 or 19-717 in SEQ ID NO:2. Anespecially preferred myosin-1F protein for use in the method consistsessentially of amino acids 1-717, 12-717 or 19-717 in SEQ ID NO:2.

[0269] In another preferred embodiment, the myosin-1F protein usedconsists essentially of a partial myosin head domain. In a preferredembodiment, such a myosin-1F protein consists essentially of an aminoacid sequence having at least about 85%, more preferably at least about90%, more preferably at least about 95%, more preferably at least about98% identity to the amino acid sequence set forth by residues 260-677 or260-691 in SEQ ID NO:2. In an especially preferred embodiment, such amyosin-1F protein consists essentially of the amino acid sequence setforth by residues 260-677 or 260-691 in SEQ ID NO:2.

[0270] In another preferred embodiment, the myosin-1F protein consistsessentially of a partial myosin head domain, an IQ domain, and a taildomain. In a preferred embodiment, such a myosin-1F protein consistsessentially of an amino acid sequence having at least about 85%, morepreferably at least about 90%, more preferably at least about 95%, morepreferably at least about 98% identity to the amino acid sequence setforth by residues 260-1098 in SEQ ID NO:2. In an especially preferredembodiment, such a myosin-1F protein consists essentially of the aminoacid sequence set forth by residues 260-1098 in SEQ ID NO:2. In anotherespecially preferred embodiment, such a myosin-1F protein consistsessentially of the amino acid sequence set forth by SEQ ID NO:4.

[0271] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, but lacks an IQ domain and atail domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 19-677, 1-677, 12-691, or 1-691 in SEQ ID NO:2.An especially preferred myosin-1F protein for use in the method consistsessentially of amino acids 19-677, 1-677, 12-691, or 1-691 in SEQ IDNO:2.

[0272] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, an IQ domain, and a taildomain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to SEQ ID NO:2. An especially preferred myosin-1F protein foruse in the method comprises SEQ ID NO:2.

[0273] By modulation of the binding of myosin-1F protein to myosin-1Fbinding partner is meant a detectable increase or decrease in binding ascompared to binding in the absence of agent, or absence of binding.

[0274] Bioactive agents that inhibit B-lymphocyte activation in theseassays are useful as immunosuppressants.

[0275] In another preferred embodiment, the methods comprise determiningthe ability of a candidate bioactive agent to modulate the activity of amyosin-1F protein.

[0276] In a preferred embodiment, the methods comprise detectingmodulation of the ATPase activity of myosin-1F in the presence ofcandidate bioactive agent, contacting the candidate bioactive agent to aB-lymphocyte, and determining lymphocyte activation in the presence ofsaid candidate agent. In a preferred embodiment, lymphocyte activationin the presence and absence of candidate agent is determined. In apreferred embodiment, an agent that normally induces lymphocyteactivation is used. Lymphocyte activation is measured in the presence,and optionally, the absence of candidate agent following contact withthe agent that normally induces lymphocyte activation. A preferredactivation agent for use with B lymphocytes is anti-IgM antibody.

[0277] In another preferred embodiment, the methods comprise detectingmodulation of the actin polymerization-inducing activity of myosin-1F inthe presence of candidate bioactive agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining lymphocyte activationin the presence of said candidate agent. In a preferred embodiment,lymphocyte activation in the presence and absence of candidate agent isdetermined. In a preferred embodiment, an agent that normally induceslymphocyte activation is used. Lymphocyte activation is measured in thepresence, and optionally, the absence of candidate agent followingcontact with the agent that normally induces lymphocyte activation. Apreferred activation agent for use with B lymphocytes is anti-IgMantibody.

[0278] In a preferred embodiment, the myosin-1F protein used in themethod comprises a myosin head domain and an IQ domain, but lacks a taildomain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to residues 1-717, 12-717, or 19-717 in SEQ ID NO:2. Anespecially preferred myosin-1F protein for use in the method consistsessentially of amino acids 1-717, 12-717, or 19-717 in SEQ ID NO:2.

[0279] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, but lacks a tail domain andan IQ domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 19-677, 1-677, 12-691, or 1-691 in SEQ ID NO:2.An especially preferred myosin-1F protein for use in the methodcomprises residues 19-677, 1-677, 12-691, or 1-691 in SEQ ID NO:2.

[0280] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, an IQ domain, and a taildomain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to SEQ ID NO:2. An especially preferred myosin-1F protein foruse in the method consists essentially of SEQ ID NO:2.

[0281] In another preferred embodiment, the methods comprise detecting achange in ATP-dependent actin binding activity of myosin-1F in thepresence of candidate bioactive agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining lymphocyte activationin the presence of said candidate agent. In a preferred embodiment,lymphocyte activation in the presence and absence of candidate agent isdetermined. In a preferred embodiment, an agent that normally induceslymphocyte activation is used. Lymphocyte activation is measured in thepresence, and optionally, the absence of candidate agent followingcontact with the agent that normally induces lymphocyte activation. Apreferred activation agent for use with B lymphocytes is anti-IgMantibody.

[0282] In a preferred embodiment, the myosin-1F protein used in themethod comprises a myosin head domain and an IQ domain, but lacks a taildomain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to residues 1-717, 12-717, or 19-717 in SEQ ID NO:2. Anespecially preferred myosin-1F protein for use in the method consistsessentially of amino acids 1-717, 12-717, or 19-717 in SEQ ID NO:2.

[0283] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, but lacks a tail domain andan IQ domain. A preferred myosin-1F protein for use in the methodconsists essentially of an amino acid sequence having at least about90%, more preferably at least about 95%, more preferably at least about98% identity to residues 19-677, 1-677, 12-691, or 1-691 in SEQ ID NO:2.An especially preferred myosin-1F protein for use in the methodcomprises residues 19-677,1-677,12-691, or 1-691 in SEQ ID NO:2.

[0284] In another preferred embodiment, the myosin-1F protein used inthe method comprises a myosin head domain, an IQ domain, and a taildomain. A preferred myosin-1F protein for use in the method consistsessentially of an amino acid sequence having at least about 90%, morepreferably at least about 95%, more preferably at least about 98%identity to SEQ ID NO:2. An especially preferred myosin-1F protein foruse in the method consists essentially of SEQ ID NO:2.

[0285] By modulation of the ATPase activity of myosin-1F is meant adetectable increase or decrease in the ability of myosin-1F to hydrolyzeATP as compared to its ability to hydrolyze ATP in the absence of agent,or loss of the ability to hydrolyze ATP.

[0286] By modulation of the actin polymerization-inducing activity ismeant a detectable increase or decrease in the ability of myosin-1F toinduce the polymerization of actin as compared to its ability to inducethe polymerization of actin in the absence of agent, or loss or theability induce the polymerization of actin.

[0287] By change in actin ATP-dependent actin binding activity is meanta detectable increase or decrease in the ability of myosin-1F to bindactin in an ATP-dependent manner as compared to its ability to do so inthe absence of agent, or loss of the ability to bind actin in anATP-dependent manner

[0288] Bioactive agents that inhibit B-lymphocyte activation in theseassays are useful as immunosuppressants.

[0289] Additional methods of screening for immunosuppressants areprovided herein.

[0290] In a preferred embodiment, the methods comprise detecting bindingof candidate agent to myosin-1F protein, contacting the candidatebioactive agent to a B-lymphocyte, and determining CD23 expression inthe presence of said candidate agent. In a preferred embodiment, CD23expression in the presence and absence of candidate agent is determined.In a preferred embodiment, an agent that normally induces CD23expression is used. Lymphocyte activation is measured in the presence,and optionally, the absence of candidate agent following contact withthe agent that normally induces CD23 expression. Preferred CD23 inducingagents are IL-4, CD40L, and the combination of IL-4 and CD40L.

[0291] Bioactive agents that inhibit CD23 induction in these assays areuseful as immunosuppressants.

[0292] In a preferred embodiment, the methods comprise detectingmodulation of the binding of myosin-1F protein to a myosin-1F bindingpartner in the presence of candidate agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining CD23 expression inthe presence of said candidate agent. In a preferred embodiment, CD23expression in the presence and absence of candidate agent is determined.In a preferred embodiment, an agent that normally induces CD23expression is used. Lymphocyte activation is measured in the presence,and optionally, the absence of candidate agent following contact withthe agent that normally induces CD23 expression. Preferred CD23 inducingagents are IL-4, CD40L, and the combination of IL-4 and CD40L.

[0293] In an especially preferred embodiment, the myosin-1F bindingpartner used in these methods is selected from the group consisting ofWASP, BLNK, WASP-IP, Bee1p, Vrp1p, calmodulin, Arp2/3 complex, Acan125,ATP and actin.

[0294] Bioactive agents that inhibit CD23 induction in these assays areuseful as immunosuppressants.

[0295] In a preferred embodiment, the methods comprise detectingmodulation of ATPase activity, or modulation of actin binding activity,or modulation of actin polymerization inducing activity of myosin-1F inthe presence of candidate bioactive agent, contacting the candidatebioactive agent to a B-lymphocyte, and determining CD23 expression inthe presence of said candidate agent. In a preferred embodiment, CD23expression in the presence and absence of candidate agent is determined.In a preferred embodiment, an agent that normally induces CD23expression is used. Lymphocyte activation is measured in the presence,and optionally, the absence of candidate agent following contact withthe agent that normally induces CD23 expression. Preferred CD23 inducingagents are IL-4, CD40L, and the combination of IL-4 and CD40L.

[0296] Bioactive agents that inhibit CD23 induction in these assays areuseful as immunosuppressants. In a preferred embodiment, the methodscomprise detecting binding of candidate agent to myosin-1F protein,contacting the candidate bioactive agent to a resting B-lymphocyte, anddetermining the level of surface Ig expression, preferably surface IgMexpression, in the cell. Particularly preferred are Ramos cells andprimary B-cells.

[0297] In a preferred embodiment, the methods comprise detectingmodulation of the binding of myosin-1F protein to a myosin-1F bindingpartner in the presence of candidate agent, contacting the candidatebioactive agent to a resting B-lymphocyte, and determining the level ofsurface Ig expression, preferably surface IgM expression, in the cell.Particularly preferred are Ramos cells and primary B-cells.

[0298] In a preferred embodiment, the methods comprise detectingmodulation of ATPase activity, or modulation of actin binding activity,or modulation of actin polymerization inducing activity of myosin-1F inthe presence of candidate bioactive agent, contacting the candidatebioactive agent to a resting B-lymphocyte, and determining the level ofsurface Ig expression, preferably surface IgM expression, in the cell.Particularly preferred are Ramos cells and primary B-cells.

[0299] Agents that decrease surface Ig expression in resting B-cells areparticularly preferred, and are useful as immunosuppressants.

[0300] In another preferred embodiment, the level of myosin-1F mRNAexpression, myosin-1F protein expression, or myosin-1F activity is usedto screen for agents that modulate the level of myosin-1F activity inB-lymphocytes. In a preferred embodiment, such agents inhibitB-lymphocyte activation and are useful as immunosuppressants.

[0301] In a preferred embodiment, candidate bioactive agents used inthese assays are small molecule chemical compounds, from about 100 toabout 1500, more preferably about 100 to about 1200, more preferablyabout 100 to about 1000, more preferably about 200 to about 500 daltons.

[0302] In a preferred embodiment, a library of candidate bioactiveagents is contacted to myosin-1F protein.

[0303] In another preferred embodiment, a library of candidate agents iscontacted to a population of cells comprising myosin-1F.

[0304] In a preferred embodiment, determining lymphocyte activation inthe methods herein comprises determining the level of expression of asurface marker which is associated with activation of a B-lymphocyte, inthe presence of candidate agent. In a preferred embodiment, the level ofsurface marker expression is determined in the presence and absence ofcandidate agent. In a preferred embodiment, the surface marker isselected from the group consisting of CD69, CD23, CD80 and CD86. In anespecially preferred embodiment, the surface marker used is CD69.

[0305] In another preferred embodiment, determining lymphocyteactivation in the methods herein comprises determining the level ofactivity of a promoter in the presence of candidate agent, whichactivity correlates with B-lymphocyte activation in the absence ofcandidate agent. In a preferred embodiment, the level of promoteractivity is determined in the presence and absence of candidate agent.In a preferred embodiment the promoter is an NFAT-responsive promoter,such as the IL-2 promoter. In an especially preferred embodiment, thepromoter is the IgH promoter.

[0306] In a preferred embodiment, determining B-lymphocyte activationinvolves measuring lymphocyte activation using a FACS machine. In apreferred embodiment, lymphocytes are sorted by FACS on the basis ofactivation.

[0307] Similarly, in a preferred embodiment, determining surface Ig,preferably surface IgM expression in a resting B-lymphocyte is doneusing a FACS machine. In a preferred embodiment, lymphocytes are sortedby FACS on the basis of surface Ig expression.

[0308] In some embodiments, the methods involve determining B-lymphocyteactivation by other means, which may also include the use of a FACSmachine. As will be appreciated, lymphocyte activation can be determinedin a number of ways. For a review of B-cell activation and methods ofmeasuring, see Clark et al., Ann. Rev. Immunol., 9:97-127, 1993, andreferences therein. It will be appreciated that mechanisms of leukocyteand platelet activation and methods for determining activation are known(see for example Kay, Immunol. Invest. 17:679-705,1988; Lukacs et. al.,Chem. Immunol. 72:102-120, 1999; Stankunas et al., Cold Spring HarborSymposia on Quant. Biol., 64: 505-516, 1999; Metcalf et. al., Physiol.Rev. 77:1033-1079, 1997; Hematol. Oncol. Clin. North Am. 4:1-26, 1990;Brass et. al., Adv. Exp. Med. Biol., 344:17-36, 1993; Brass et. al.,Thromb. Haemost., 70:217-223, 1993; Cellular and Molecular Immunology,Abbas et. al., W. B. Saunders, ISBN 0-7216-3032-4, Chapters 7, 9, 12,and 14). Particularly relevant are the methods disclosed by Holland etal., J. Exp. Med., 194:1263-1276, 2001, expressly incorporated herein byreference.

[0309] In some embodiments, indicators of lymphocyte activation areused. There are a number of parameters that may be evaluated or assayedto determine lymphocyte activation, including, but not limited to, IgHpromoter activity, calcium flux, NFAT activity, Ig secretion, IgG andIgM production, lymphocyte proliferation, expression cell surfacemarkers correlated with lymphocyte activation, cytokine production,intracellular calcium levels, release of calcium from intracellularstores, amount of SYK protein, level of SYK protein ubiquitination, SYKprotein tyrosine kinase activity, and IL-2 expression. These parametersmay be assayed and used as indicators to evaluate the effect ofcandidate drug agents on lymphocyte activation. In this manner, rapid,accurate screening of candidate agents may be performed to identifyagents that modulate lymphocyte activation.

[0310] By a “population of cells” or “library of cells” herein is meantat least two cells, with at least about 10³ being preferred, at leastabout 10⁶ being particularly preferred, and at least about 10⁸ to 10⁹being especially preferred. The population or sample can contain amixture of different cell types from either primary or secondarycultures although samples containing only a single cell type arepreferred, for example, the sample can be from a cell line, particularlytumor cell lines, as outlined below. In a preferred embodiment, cellsthat are replicating or proliferating are used; this may allow the useof retroviral vectors for the introduction of candidate bioactiveagents. Alternatively, non-replicating cells may be used, and othervectors (such as adenovirus and lentivirus vectors) can be used. Inaddition, although not required, the cells are compatible with dyes andantibodies.

[0311] Preferred cell types for use in the invention include, but arenot limited to, mammalian cells, including animal (rodents, includingmice, rats, hamsters and gerbils), primates, and human cells,particularly including tumor cells of all types, including breast, skin,lung, cervix, colonrectal, leukemia, brain, etc. More preferable celltypes include the Ig(+) and IgM secreting B-cell lines CL-01, LA350,BJAB, and CA46. Primary cells are also preferred, including peripheralblood lymphocytes (PBLs) and peripheral blood mononuclear cells (PBMCs).Ramos cells (B-cell cell line) are also preferred.

[0312] In the methods provided herein requiring the use ofB-lymphocytes, B-lymphocyte-like cells or B-lymphocyte cell lines, suchas those described above, or primary B-lymphocytes may be used.

[0313] Preferred cell surface markers useful as indicators ofB-lymphocyte activation in the methods herein exhibit low backgroundexpression in the absence of lymphocyte activation. Especially preferredcell surface markers include CD69, CD23, CD80, CD86. CD69 and CD23 areespecially preferred.

[0314] Agents that recognize such surface molecules (e.g. antibodies)can be used as an affinity ligand, and attached to a solid support suchas a bead, a surface, etc., and used to pull out B-cells that areundergoing activation. Similarly, these agents can be coupled to afluorescent dye such as PerCP, and then assayed using a FACS machine,and cells may be optionally sorted on this basis.

[0315] FACS analysis can be used in conjunction with antibodiesrecognizing lymphocyte surface markers that are correlated withlymphocyte activation. A FACS machine is used to analyze, and optionallysort cells based on the expression of these markers to detectunstimulated and stimulated lymphocytes. In a preferred embodiment,sorted lymphocytes are used to retrieve candidate bioactive agentsintroduced thereto.

[0316] In a preferred embodiment, IgH promoter activity and NFATactivity are measured using lymphocyte clones comprising an IgH promoteror an NFAT-responsive promoter (such as IL-2 promoter) operably linkedto a reporter gene. For example, a surface Ig(+), IgM secreting B-cellline such as the BJAB, CL-01, CA46, or LA350 cell line is transfectedwith a construct comprising GFP/2a/TK fusion under the control of an IgHpromoter, Eμ and 3′α enhancer elements. Stable transfectants (referredto herein as immunoglobulin heavy chain reporter cell lines) areselected and maintained in gancyclovir. Preferred immunoglobulin heavychain reporter cell lines for use in the present invention exhibit lowbackground GFP expression and strong basal activity and/or inducibleactivity in the presence of positive control. Such cell lines can begenerated with the use of retroviral constructs.

[0317] Release of calcium from intracellular calcium stores may beassayed using membrane permeant vital calcium sensing fluorescent dyes,as are well known in the art. For example, see Calcium Green™, CalciumOrange™, from Molecular Probes, Eugene, Oreg., catalog numbers C-3010,C-3013, for example.

[0318] A preferred embodiment utilizes a cell proliferation assay. Forexample, B-cells proliferate when activated. By “proliferation assay”herein is meant an assay that allows the determination that a cellpopulation is either proliferating, i.e. replicating, or notproliferating.

[0319] In a preferred embodiment, the proliferation assay is a dyeinclusion assay. A dye inclusion assay relies on dilution effects todistinguish between proliferating and non-proliferating cells. Briefly,a dye (generally a fluorescent dye as outlined below) is introduced tocells and taken up by the cells. Once taken up, the dye is trapped inthe cell, and does not diffuse out. As the cell population divides, thedye is proportionally diluted. That is, after the introduction of theinclusion dye, the cells are allowed to incubate for some period oftime; cells that lose fluorescence over time are dividing, and the cellsthat remain fluorescent are arrested in a non-growth phase.

[0320] The rate of loss of fluorescence is indicative of the rate ofproliferation. An increase in proliferation rate above that ofunstimulated cells is indicative of B-cell activation.

[0321] These methods may also be applied to platelets.

[0322] Generally, the introduction of the inclusion dye may be done inone of two ways. Either the dye cannot passively enter the cells (e.g.it is charged), and the cells must be treated to take up the dye; forexample through the use of an electric pulse. Alternatively, the dye canpassively enter the cells, but once taken up, it is modified such thatit cannot diffuse out of the cells. For example, enzymatic modificationof the inclusion dye may render it charged, and thus unable to diffuseout of the cells. For example, the Molecular Probes CellTracker™ dyesare fluorescent chloromethyl derivatives that freely diffuse into cells,and then glutathione S-transferase-mediated reaction produces membraneimpermeant dyes.

[0323] Suitable inclusion dyes include, but are not limited to, theMolecular Probes line of CellTracker™ dyes, including, but not limitedto CellTracker™ Blue, CellTracker™ Yellow-Green, CellTracker™ Green,CellTracker™ Orange, PKH26 (Sigma), and others known in the art; see theMolecular Probes Handbook; chapter 15 in particular.

[0324] In general, inclusion dyes are provided to the cells at aconcentration ranging from about 100 ng/ml to about 5 μg/ml, with fromabout 500 ng/ml to about 1 μg/ml being preferred. A wash step may or maynot be used. In a preferred embodiment, a candidate bioactive agent iscombined with the cells as described herein. The cells and the inclusiondye are incubated for some period of time, to allow cell division andthus dye dilution.

[0325] Without being bound by theory, it is recognized herein thatmyosin-1F proteins are involved in the regulation of signal transductionin B-lymphocytes and platelets. Particularly, myosin-1F proteins arerecognized herein as being critical regulators of B-cell activation aswell as platelet proliferation. As discussed above, the activation ofspecific signaling pathways in lymphocytes determines the quality,magnitude, and duration of immune responses. In transplantation, acuteand chronic inflammatory diseases, and autoimmunity, it is thesepathways that are responsible for the induction, maintenance andexacerbation of undesirable lymphocyte responses.

[0326] Accordingly, in one aspect, the invention provides compositionsand methods for the treatment of B-lymphocyte activation disorders andplatelet activation or proliferation disorders, as described below.

[0327] In a preferred embodiment, the present invention providesmyosin-1F proteins and nucleic acids, as well as agents that bind tothem and/or modulate their activity, including and preferably smallmolecule chemical compositions as discussed herein, which are useful inthe treatment of acute and chronic inflammatory diseases and autoimmunediseases, as well as in the treatment of a host receiving a transplant.Among these diseases are those listed in FIG. 5.

[0328] In another preferred embodiment, the present invention providesmyosin-1F proteins and nucleic acids, as well as agents that bind tothem and/or modulate their activity, including and preferably smallmolecule chemical compositions as discussed herein, which are useful inthe treatment of physiological states that are characterized by or leadto the presentation of some or all symptoms characteristic of acuteinflammatory disease, chronic inflammatory disease, autoimmune disease,or response to transplantation.

[0329] It will be understood that these diseases and states may or maynot be associated with altered myosin-1F activity. That is, myosin-1Fcompositions (proteins, nucleic acids, anti-myosin-1F antibodies,agonists, antagonists) find use in the prevention and/or treatment ofdiseases and states which do not have myosin-1F dysregulation ordysfunction as a molecular basis, but still involve lymphocyteactivation or platelet proliferation. That is, a disease or state neednot be associated with myosin-1F activity for the present compositionsand methods to be useful in preventing or treating it. Many autoimmunediseases fall into this category.

[0330] In another preferred embodiment, the present invention providesmyosin-1F proteins and nucleic acids, as well as agents that bind tothem and/or modulate their activity, including and preferably smallmolecule chemical compositions as discussed herein, which are useful asprophylactics for the prevention of acute inflammatory disease, chronicinflammatory disease, autoimmune disease, and response totransplantation.

[0331] In a preferred embodiment, the present invention providesmyosin-1F proteins and nucleic acids, as well as agents that bind tothem and/or modulate their activity, including and preferably smallmolecule chemical compositions as discussed herein, which are useful asprophylactics for the prevention of physiological states that arecharacterized by or lead to the presentation of some or all symptomscharacteristic of acute inflammatory disease, chronic inflammatorydisease, autoimmune disease, or response to transplantation.

[0332] In a preferred embodiment, myosin-1F proteins and nucleic acidsprovided herein are useful for the modulation of antigenreceptor-induced B-lymphocyte activation, as characterized by theinduction of CD69 and other markers of activation.

[0333] In an especially preferred embodiment, myosin-1F proteins andnucleic acids provided herein are useful for the modulation ofimmunoglobulin production by B-lymphocytes that normally results fromBCR activation by antigen.

[0334] Particularly useful for these purposes is a myosin-1F variantprotein comprising an SH3 domain having a point mutation which disruptsbinding to a myosin-1F binding partner. Another myosin-1F proteinparticularly useful for this purpose is a variant myosin-1F proteincomprising a myosin head domain having a point mutation in theATP-binding domain which is incapable of hydrolyzing ATP. Anothermyosin-1F protein particularly useful for this purpose consistsessentially of amino acids 617-1098 in SEQ ID NO:2. Another myosin-1Fprotein particularly useful for this purpose consists essentially of apolynucleotide encoded by SEQ ID NO:5.

[0335] Without being bound by theory, myosin-1F proteins, beingmodulators of signal transduction in B-lymphocytes, particularly signaltransduction events underlying B-lymphocyte activation, are involved inthe regulation of proliferation of B-lymphocytes, and have utility asmodulators of lymphocyte proliferation. Further, disorders associatedwith myosin-1F dysfunction or dysregulation include lymphocyteproliferation disorders, such as B-cell leukemias, lymphomas, andWiskott-Aldrich syndrome.

[0336] Accordingly, in a preferred embodiment, the present inventionprovides myosin-1F proteins and nucleic acids, as well as agents capableof binding to them or modulating their activity, including andpreferably small molecule chemical compositions as discussed herein,which are useful in the treatment of disorders involving B-cellproliferation, including leukemias, lymphomas, and Wiskott-Aldrichsyndrome.

[0337] Without being bound by theory, myosin-1F proteins, beingmodulators of signal transduction in platelets, are involved in theregulation of proliferation of platelets, and have utility as modulatorsof platelet proliferation. Further, disorders associated with myosin-1Fdysfunction or dysregulation include platelet proliferation disorders,such as Wiskott-Aldrich syndrome.

[0338] It is recognized in the art that signaling pathways involved inthe regulation of cell proliferation frequently participate in, directlyor indirectly, the regulation of cell survival and programmed celldeath. It is further recognized in the art that the dysregulation ofmechanisms of programmed cell death can lead to cancer, particularly inlymphocytes. For example, overexpression of Bcl-2, which promotes cellsurvival through the inhibition of apoptotic processes, is thought to beresponsible for the survival of excessive numbers of lymphocytes in aform of lymphoma (Reed et al., Science, 236:1295-1299, 1987; Tsujimotoet al., Science, 228:1440-1443, 1985).

[0339] Accordingly, the present invention provides myosin-1F proteinsand nucleic acids, as well as agents capable of binding to them and/ormodulating their activity, including and preferably small moleculechemical compositions as discussed herein, which are useful in thetreatment of disorders involving B-cell survival and programmed celldeath, including cancer.

[0340] The proteins and nucleic acids provided herein can also be usedfor screening purposes wherein the protein-protein interactions of themyosin-1F proteins can be identified. Genetic systems have beendescribed to detect protein-protein interactions. The first work wasdone in yeast systems, namely the “yeast two-hybrid” system. The basicsystem requires a protein-protein interaction in order to turn ontranscription of a reporter gene. Subsequent work was done in mammaliancells. See Fields et al., Nature 340:245 (1989); Vasavada et al., PNASUSA 88:10686 (1991); Fearon et al., PNAS USA 89:7958 (1992); Dang etal., Mol. Cell. Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578(1991); and U.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490,and 5,637,463. A preferred system is described in Ser. No. 09/050,863,filed Mar. 30, 1998 and Ser. No. 09/359,081 filed Jul. 22, 1999,entitled “Mammalian Protein Interaction Cloning System”. For use inconjunction with these systems, a particularly useful shuttle vector isdescribed in Ser. No. 09/133,944, filed Aug. 14, 1998, entitled “ShuttleVectors”.

[0341] In general, two nucleic acids are transformed into a cell, whereone is a “bait” such as the gene encoding a myosin-1F protein or aportion thereof, and the other encodes a test candidate. Only if the twoexpression products bind to one another will an indicator, such as afluorescent protein, be expressed. Expression of the indicator indicateswhen a test candidate binds to the myosin-1F protein, and identifies thecandidate as being part of a B-cell or platelet myosin-1F signalingpathway. A test candidate so identified may then be used as bait toidentify binding proteins that are also identified as being part of aB-cell or platelet myosin-1F signaling pathway. Additionally, myosin-1Fproteins may be used to identify new baits, or agents that bind tomyosin-1F proteins. Additionally, the two-hybrid system can be usedwherein a test candidate is added in addition to the bait and themyosin-1F protein encoding nucleic acids to determine agents whichinterfere with the binding of bait to the myosin-1F protein.

[0342] In one embodiment, a mammalian two-hybrid system is preferred.Mammalian systems provide post-translational modifications of proteinswhich may contribute significantly to their ability to interact. Inaddition, a mammalian two-hybrid system can be used in a wide variety ofmammalian cell types to mimic the regulation, induction, processing,etc. of specific proteins within a particular cell type. For example,proteins involved in a disease state (i.e., cancer, apoptosis relateddisorders) could be tested in the relevant disease cells. Similarly, fortesting of random proteins, assaying them under the relevant cellularconditions will give the highest positive results. Furthermore, themammalian cells can be tested under a variety of experimental conditionsthat may affect intracellular protein-protein interactions, such as inthe presence of hormones, drugs, growth factors and cytokines,radiation, chemotherapeutics, cellular and chemical stimuli, etc., thatmay contribute to conditions which can affect protein-proteininteractions.

[0343] Assays involving binding such as the two-hybrid system may takeinto account non-specific binding proteins (NSB).

[0344] Expression in various cell types, and assays for myosin-1Factivity are described above. The activity assays can be performed toconfirm the activity of myosin-1F proteins which have already beenidentified by their sequence identity/similarity to myosin-1F (SEQ IDNO:2), as well as to further confirm the activity of lead compoundsidentified as modulators of myosin-1F activity.

[0345] The components provided herein for the assays provided herein mayalso be combined to form kits. The kits can be based on the use ofmyosin-1F proteins and/or nucleic acids encoding myosin-1F proteins. Inone embodiment, other components are provided in the kit. Suchcomponents include one or more of packaging, instructions, antibodies,and labels. Additional assays such as those used in diagnostics arefurther described below.

[0346] Bioactive agents may be identified by the methods providedherein. Compounds with pharmacological activity are able to enhance orinterfere with the activity of the myosin-1F protein. The compoundshaving the desired pharmacological activity may be administered in aphysiologically acceptable carrier to a host, as further describedbelow.

[0347] The present discovery relating to the role of myosin-1F proteinsB-lymphocytes thus provides methods for inducing or preventingB-lymphocyte activation and platelet proliferation. In a preferredembodiment, the myosin-1F proteins, and particularly myosin-1F proteinfragments, are useful in the study or treatment of conditions whichinvolve dysfunction or dysregulation of myosin-1F protein activity, i.e.to diagnose, treat or prevent myosin-1F associated disorders. “Myosin-1Fassociated disorders” or “disease states” or “physiological statesassociated with myosin-1F dysfunction or dysregulation” includeconditions involving insufficient, excessive, and inappropriatemyosin-1F activity. Among these disorders are B-lymphocyte activationdisorders, and platelet proliferation disorders.

[0348] Thus, in one embodiment, methods for regulating B-lymphocyteactivation in cells or organisms are provided. In one embodiment, themethods comprise administering to a cell or individual, a myosin-1Fprotein in a therapeutic amount. Alternatively, an anti-myosin-1Fantibody that reduces or eliminates the biological activity of theendogenous myosin-1F protein is administered. Particularly preferred areintrabodies, which are useful for the inhibition of intracellularmyosin-1F protein in situ. The use of intrabodies is known in the art,for example, see Marasco, Curr. Top. Microbiol. Immunol. 260:247-270,2001. Alternatively and preferably a myosin-1F dominant negative proteinvariant is administered. In another preferred embodiment, a bioactiveagent as identified by the methods provided herein is administered. In afurther preferred embodiment, a small molecule chemical compositionwhich inhibits myosin-1F activity is administered. Alternatively, themethods comprise administering to a cell or individual a recombinantnucleic acid encoding a myosin-1F protein. In one embodiment, nucleicacid encoding a myosin-1F dominant negative variant protein isadministered. In another embodiment, a myosin-1F antisense nucleic acidis administered. In another embodiment, a myosin-1F RNAi isadministered. RNAi's are well known in the art, for example, see Caplan,Trends in Biotechnology, 20: 49-51, 2002.

[0349] In one embodiment, the activity of myosin-1F is increased. Aswill be appreciated by those in the art, this may be accomplished in anynumber of ways. In a preferred embodiment, the activity of myosin-1F isincreased by increasing the amount of myosin-1F in the cell, for exampleby overexpressing the endogenous myosin-1F or by administering a geneencoding a myosin-1F protein, using known gene-therapy techniques, forexample. In a preferred embodiment, the gene therapy techniques includethe incorporation of the exogenous gene using enhanced homologousrecombination (EHR), for example as described in PCT/US93/03868, herebyincorporated by reference in its entirety. Alternatively, myosin-1Factivity may be increased by administering an agent determined toincrease myosin-1F activity or expression by the methods providedherein.

[0350] In one embodiment, the activity of myosin-1F is decreased. Aswill be appreciated by those in the art, this may be accomplished in anynumber of ways. In a preferred embodiment, the activity of myosin-1F isdecreased by decreasing the amount of myosin-1F mRNA in the cell, forexample by expressing myosin-1F antisense RNA. Double stranded nucleicacids for use in RNA interference (see Caplan, Trends in Biotechnology,20: 49-51, 2002) are also preferred for this purpose. Alternatively,endogenous myosin-1F activity is decreased by administering a dominantnegative myosin-1F protein or a gene encoding a dominant negativemyosin-1F protein. Alternatively, endogenous myosin-1F activity isdecreased by administering anti-myosin-1F antibody or a gene encodinganti-myosin-1F antibody or an epitope recognizing portion thereof.Particularly preferred are intrabodies, which are useful for theinhibition of intracellular myosin-1F protein in situ. Knowngene-therapy techniques may be used to administer these agents. In apreferred embodiment, the gene therapy techniques involve incorporationof the exogenous gene into the host genome using enhanced homologousrecombination (EHR), for example as described in PCT/US93/03868, herebyincorporated by reference in its entirety. Alternatively, myosin-1Factivity may be decreased by administering an agent determined todecrease myosin-1F activity or expression by the methods providedherein.

[0351] It appears that myosin-1F protein is an important protein inB-lymphocyte activation and platelet proliferation. Accordingly,disorders based on mutant or variant myosin-1F genes may be determined.In one embodiment, the invention provides methods for identifying cellscontaining variant myosin-1F genes comprising determining all or part ofthe sequence of at least one endogenous myosin-1F gene in a cell. Aswill be appreciated by those in the art, this may be done using anynumber of sequencing techniques. In a preferred embodiment, theinvention provides methods of identifying the myosin-1F genotype of anindividual comprising determining all or part of the sequence of atleast one myosin-1F gene of the individual. This is generally done in atleast one tissue of the individual, and may include the evaluation of anumber of tissues or different samples of the same tissue. The methodmay include comparing the sequence of the sequenced myosin-1F gene to aknown myosin-1F gene, i.e. a wild-type gene.

[0352] The sequence of all or part of the myosin-1F gene can then becompared to the sequence of a known myosin-1F gene to determine if anydifferences exist. This can be done using any number of known sequenceidentity programs, such as Bestfit, etc. In a preferred embodiment, thepresence of a difference in the sequence between the myosin-1F gene ofthe patient and the known myosin-1F gene is indicative of a diseasestate or a propensity for a disease state, particularly a B-lymphocyteactivation disorder or a platelet proliferation disorder.

[0353] In one embodiment, the invention provides methods for diagnosinga myosin-1F related condition in an individual. The methods comprisemeasuring the activity of myosin-1F in a tissue from the individual orpatient, which may include a measurement of the amount or specificactivity of a myosin-1F protein. This activity is compared to theactivity of myosin-1F from either an unaffected second individual orfrom an unaffected tissue from the first individual. When theseactivities are different, the first individual may be at risk for amyosin-1F associated disorder. In this way, for example, monitoring ofvarious disease conditions may be done by monitoring the absolutemyosin-1F activity in a sample or the specific activity of a myosin-1Fprotein from a sample. Similarly, activity levels may correlate withprognosis.

[0354] In a preferred embodiment, myosin-1F activity levels aredetermined in B-lymphocytes of an affected individual. In anotherpreferred embodiment, myosin-1F activity levels are determined inplatelets of an affected individual.

[0355] In one aspect, the expression levels of myosin-1F genes (encodingmyosin-1F proteins) are determined in different patient samples or cellsfor which either diagnostic or prognostic information is desired. Geneexpression monitoring is done on genes encoding myosin-1F proteins. Inone aspect, the expression levels of myosin-1F genes are determined fordifferent cellular states, such as normal cells and activated cells. Bycomparing myosin-1F gene expression levels in cells in different states,information including both up- and down-regulation of myosin-1F genes isobtained, which can be used in a number of ways. For example, theevaluation of a particular treatment regime may be evaluated: does achemotherapeutic drug act to improve the long-term prognosis in aparticular patient. Similarly, diagnosis may be done or confirmed bycomparing patient samples. Furthermore, these gene expression levelsallow screening of drug candidates with an eye to mimicking or alteringa particular expression level. This may be done by making biochipscomprising probes that determine the presence of myosin-1F genes, whichbiochips can be used in these screens. These methods can also be done onthe protein basis; that is, myosin-1F protein expression levels can beevaluated for diagnostic and prognostic purposes or to screen candidateagents for their effects on myosin-1F protein expression. ELISA methods,and array-based protein detection methods are know to those skilled inthe art.

[0356] In a preferred embodiment, myosin-1F expression levels aredetermined in B-lymphocytes in the presence of candidate agents. Thisdetermination is done to screen for agents capable of modulatingmyosin-1F expression, which find use as immunosuppressants and as agentsfor the inhibition of B-lymphocyte activation.

[0357] In a preferred embodiment, nucleic acid probes to myosin-1Fnucleic acids and their complements are made. The nucleic acid probesare designed to be substantially complementary to myosin-1F nucleicacids, i.e., the target sequence, such that hybridization of the targetsequence and the probe occurs. As outlined below, this complementarityneed not be perfect; there may be any number of base pair mismatcheswhich will interfere with hybridization between the target sequence andthe single stranded nucleic acids of the present invention. However, ifthe number of mismatches is so great that no hybridization can occurunder even the least stringent of hybridization conditions, the sequenceis not a complementary target sequence. Thus, by “substantiallycomplementary” herein is meant that the probes are sufficientlycomplementary to the target sequences to hybridize under normal reactionconditions, particularly high stringency conditions, as outlined herein.

[0358] A “nucleic acid probe” is generally single stranded but can bepartially single and partially double stranded. The strandedness of theprobe is dictated by the structure, composition, and properties of thetarget sequence. In general, the nucleic acid probes range from about 8to about 100 bases long, with from about 10 to about 80 bases beingpreferred, and from about 30 to about 50 bases being particularlypreferred. In some embodiments, much longer nucleic acids can be used,up to hundreds of bases (e.g., whole genes).

[0359] As will be appreciated by those in the art, nucleic acids can beattached or immobilized to a solid support in a wide variety of ways. By“immobilized” and grammatical equivalents herein is meant theassociation or binding between the nucleic acid probe and the solidsupport is sufficient to be stable under the conditions of binding,washing, analysis, and removal. The binding can be covalent ornon-covalent. By “non-covalent binding” and grammatical equivalentsherein is meant one or more of either electrostatic, hydrophilic, andhydrophobic interactions. Included in non-covalent binding is thecovalent attachment of a molecule, such as, streptavidin to the supportand the non-covalent binding of the biotinylated probe to thestreptavidin. By “covalent binding” and grammatical equivalents hereinis meant that the two moieties, the solid support and the probe, areattached by at least one bond, including sigma bonds, pi bonds andcoordination bonds. Covalent bonds can be formed directly between theprobe and the solid support or can be formed by a cross linker or byinclusion of a specific reactive group on either the solid support orthe probe or both molecules. Immobilization may also involve acombination of covalent and non-covalent interactions.

[0360] In general, the probes are attached to the biochip in a widevariety of ways, as will be appreciated by those in the art. Asdescribed herein, the nucleic acids can either be synthesized first,with subsequent attachment to the biochip, or can be directlysynthesized on the biochip.

[0361] The biochip comprises a suitable solid substrate. By “substrate”or “solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of the nucleic acid probesand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates are very large,and include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, etc. In general, the substrates allow optical detection and donot appreciably show fluorescence.

[0362] In a preferred embodiment, the surface of the biochip and theprobe may be derivatized with chemical functional groups for subsequentattachment of the two. Thus, for example, the biochip is derivatizedwith a chemical functional group including, but not limited to, aminogroups, carboxy groups, oxo groups and thiol groups, with amino groupsbeing particularly preferred. Using these functional groups, the probescan be attached using functional groups on the probes. For example,nucleic acids containing amino groups can be attached to surfacescomprising amino groups, for example using linkers as are known in theart; for example, homo-or hetero-bifunctional linkers as are well known(see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference). Inaddition, in some cases, additional linkers, such as alkyl groups(including substituted and heteroalkyl groups) may be used.

[0363] In this embodiment, oligonucleotides, corresponding to thenucleic acid probe, are synthesized as is known in the art, and thenattached to the surface of the solid support. As will be appreciated bythose skilled in the art, either the 5′ or 3′ terminus may be attachedto the solid support, or attachment may be via an internal nucleoside.

[0364] In an additional embodiment, the immobilization to the solidsupport may be very strong, yet non-covalent. For example, biotinylatedoligonucleotides can be made, which bind to surfaces covalently coatedwith streptavidin, resulting in attachment.

[0365] Alternatively, the oligonucleotides may be synthesized on thesurface, as is known in the art. For example, photoactivation techniquesutilizing photopolymerization compounds and techniques are used. In apreferred embodiment, the nucleic acids can be synthesized in situ,using well known photolithographic techniques, such as those describedin WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; andreferences cited within, all of which are expressly incorporated byreference; these methods of attachment form the basis of the AffimetrixGeneChip™technology.

[0366] “Differential expression,” or grammatical equivalents as usedherein, refers to both qualitative as well as quantitative differencesin the genes' temporal and/or cellular expression patterns within andamong the cells. Thus, a differentially expressed gene can qualitativelyhave its expression altered, including an activation or inactivation,in, for example, a normal versus an apoptotic cell. That is, genes maybe turned on or turned off in a particular state, relative to anotherstate. As is apparent to the skilled artisan, any comparison of two ormore states can be made. Such a qualitatively regulated gene willexhibit an expression pattern within a state or cell type which isdetectable by standard techniques in one such state or cell type, but isnot detectable in both. Alternatively, the determination is quantitativein that expression is increased or decreased; that is, the expression ofthe gene is either upregulated, resulting in an increased amount oftranscript, or downregulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques, such as byuse of Affymetrix GeneChip™ expression arrays, Lockhart, NatureBiotechnology 14:1675-1680 (1996), hereby expressly incorporated byreference. Other techniques include, but are not limited to,quantitative reverse transcriptase PCR, Northern analysis and RNaseprotection.

[0367] Though discussed above with respect to transcripts, it will beappreciated by those in the art that this may be done by evaluation ateither the gene transcript, or the protein level; that is, the amount ofgene expression may be monitored using nucleic acid probes to the DNA orRNA gene transcript, and the quantification of gene expression levels,or, alternatively, the final gene product itself (protein) can bemonitored, for example through the use of antibodies to the myosin-1Fprotein and standard immunoassays (ELISAs, etc.) or other techniques,including mass spectroscopy assays, 2D gel electrophoresis assays, etc.

[0368] In another method detection of the mRNA is performed in situ. Inthis method permeabilized cells or tissue samples are contacted with adetectably labeled nucleic acid probe for sufficient time to allow theprobe to hybridize with the target mRNA. Following washing to remove thenon-specifically bound probe, the label is detected. For example adigoxygenin labeled riboprobe (RNA probe) that is complementary to themRNA encoding a Myosin-1F protein is detected by binding the digoxygeninwith an anti-digoxygenin secondary antibody and exposed to nitro bluetetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.

[0369] In another preferred method, expression of myosin-1F protein isdetermined using in situ imaging techniques employing antibodies tomyosin-1F proteins. In this method cells are contacted with from one tomany antibodies to the myosin-1F protein(s). Following washing to removenon-specific antibody binding, the presence of the antibody orantibodies is detected. In one embodiment the antibody is detected byincubating with a secondary antibody that contains a detectable label.In another method the primary antibody to the myosin-1F protein(s)contains a detectable label. In another preferred embodiment each one ofmultiple primary antibodies contains a distinct and detectable label.This method finds particular use in simultaneous screening for aplurality of myosin-1F proteins. The label may be detected in afluorometer which has the ability to detect and distinguish emissions ofdifferent wavelengths. Labels may be detected using a fluorescencemicroscope which has multiple fluorescence channels. In addition, afluorescence activated cell sorter (FACS) can be used in this method. Aswill be appreciated by one of ordinary skill in the art, numerous otherhistological imaging techniques are useful in the invention and theantibodies can also be used in ELISA, immunoblotting (Western blotting),immunoprecipitation, BIACORE technology, and the like.

[0370] In one embodiment, the myosin-1F proteins of the presentinvention may be used to generate polyclonal and monoclonal antibodiesto myosin-1F proteins, which are useful as described herein. Similarly,the myosin-1F proteins can be coupled, using standard technology, toaffinity chromatography columns. These columns may then be used topurify myosin-1F antibodies. In a preferred embodiment, the antibodiesare generated to epitopes unique to the myosin-1F protein; that is, theantibodies show little or no cross-reactivity to other proteins. Theseantibodies find use in a number of applications. For example, themyosin-1F antibodies may be coupled to standard affinity chromatographycolumns and used to purify myosin-1F proteins as further describedbelow. The antibodies may also be used as blocking polypeptides, asoutlined above, since they will specifically bind to the myosin-1Fprotein.

[0371] The anti-myosin-1F protein antibodies may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include themyosin-1F protein or a fusion protein thereof. It may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Examples of such immunogenic proteinsinclude but are not limited to keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, and soybean trypsin inhibitor. Examples ofadjuvants which may be employed include Freund's complete adjuvant andMPL-TDM adjuvant (monophosphoryl Lipid a, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

[0372] The anti-myosin-1F protein antibodies may, alternatively, bemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature 256:495 (1975). In a hybridoma method, a mouse, hamster, or otherappropriate host animal, is typically immunized with an immunizing agentto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

[0373] The immunizing agent will typically include the Myosin-1F proteinor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0374] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0375] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst myosin-1F protein. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0376] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0377] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein a-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0378] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0379] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0380] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0381] Alternatively, intrabodies may be prepared that are capable ofbinding to myosin-1F intracellularly. Wirtz et al, Prot. Sci.8(11):2245-50 (1999); Ohage et al. J. Mol. Biol. 291(5):1129-34 andOhage et al. J. Biol. Chem. 291(5): 1119-28 (1999), the disclosures ofwhich are expressly incorporated by reference herein. Preferably suchintrabodies are lipid soluble and lack a constant region. Intrabodiesare particularly useful for the treatment of B-lymphocyte activationdisorders, including those associated with myosin-1F dysregulation ordysfunction.

[0382] The anti-myosin-1F protein antibodies of the invention mayfurther comprise humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op Struct. Biol2:593-596 (1992)].

[0383] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0384] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Bio. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0385] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the myosin-1F protein, the other one is for anyother antigen, and preferably for a cell-surface protein or receptor orreceptor subunit, preferably for a protein on the surface of adysregulated or dysfunctional B-lymphocyte or platelet.

[0386] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0387] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0388] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0389] The anti-myosin-1F protein antibodies of the invention havevarious utilities. For example, anti-myosin-1F protein antibodies may beused in diagnostic assays for a myosin-1F protein, e.g., detecting itsexpression in specific cells, tissues, or serum. Various diagnosticassay techniques known in the art may be used, such as competitivebinding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: a Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

[0390] Anti-myosin-1F protein antibodies also are useful for theaffinity purification of myosin-1F protein from recombinant cell cultureor natural sources. In this process, the antibodies against myosin-1Fprotein are immobilized on a suitable support, such a Sephadex resin orfilter paper, using methods well known in the art. The immobilizedantibody then is contacted with a sample containing the myosin-1Fprotein to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the myosin-1F protein, which is bound to the immobilizedantibody. Finally, the support is washed with another suitable solventthat will release the myosin-1F protein from the antibody.

[0391] The anti-myosin-1F protein antibodies may also be used intreatment. In one embodiment, the genes encoding the antibodies areprovided, such that the antibodies bind to and modulate the myosin-1Fprotein within the cell.

[0392] In one embodiment, anti-myosin-1F antibodies provided herein arecapable of reducing or eliminating myosin-1F bioactivity. Theseantibodies are sometimes referred to herein as function-blocking orfunction-inhibiting antibodies.

[0393] In another embodiment, anti-myosin-1F antibodies provided hereinare capable of increasing or enhancing myosin-1F bioactivity. Theseantibodies are sometimes referred to herein as function-activatingantibodies.

[0394] In one embodiment, a therapeutically effective dose of amyosin-1F protein, agonist or antagonist is administered to a patient.By “therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. As is known in the art, adjustmentsfor myosin-1F protein degradation, systemic versus localized delivery,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

[0395] A “patient” for the purposes of the present invention includesboth humans and other animals, particularly mammals. Thus the methodsare applicable to both human therapy and veterinary applications. In thepreferred embodiment the patient is a mammal, and in the most preferredembodiment the patient is human.

[0396] The administration of the myosin-1F protein, agonist orantagonist of the present invention can be done in a variety of ways,including, but not limited to, orally, subcutaneously, intravenously,intranasally, transdermally, intraperitoneally, intramuscularly,intrapulmonary, vaginally, rectally, or intraocularly. In someinstances, for example, in the treatment of wounds and inflammation, thecomposition may be directly applied as a solution or spray. Dependingupon the manner of introduction, the compounds may be formulated in avariety of ways. The concentration of therapeutically active compound inthe formulation may vary from about 0.1-100% wt.

[0397] The pharmaceutical compositions of the present invention comprisea myosin-1F protein, agonist or antagonist (including antibodies andbioactive agents as described herein, most preferably small moleculechemical compositions as described herein) in a form suitable foradministration to a patient. In the preferred embodiment, thepharmaceutical compositions are in a water soluble form, such as beingpresent as pharmaceutically acceptable salts, which is meant to includeboth acid and base addition salts. “Pharmaceutically acceptable acidaddition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferred are the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine.

[0398] The pharmaceutical compositions may also include one or more ofthe following: carrier proteins such as serum albumin; buffers; fillerssuch as microcrystalline cellulose, lactose, corn and other starches;binding agents; sweeteners and other flavoring agents; coloring agents;and polyethylene glycol. Additives are well known in the art, and areused in a variety of formulations.

[0399] Combinations of the compositions may be administered. Moreover,the compositions may be administered in combination with othertherapeutics, including growth factors or chemotherapeutics and/orradiation. Targeting agents (i.e. ligands for receptors on cancer cells)may also be combined with the compositions provided herein.

[0400] In one embodiment provided herein, the antibodies are used forimmunotherapy, thus, methods of immunotherapy are provided. By“immunotherapy” is meant treatment of myosin-1F protein relateddisorders with an antibody raised against a myosin-1F protein.Immunotherapy may also be used to treat lymphocyte disorders notassociated with myosin-1F, but treatable by myosin-1F modulation. Asused herein, immunotherapy can be passive or active. Passiveimmunotherapy, as defined herein, is the passive transfer of antibody toa recipient (patient). Active immunization is the induction of antibodyand/or T-cell responses in a recipient (patient). Induction of an immuneresponse can be the consequence of providing the recipient with amyosin-1F protein antigen to which antibodies are raised. As appreciatedby one of ordinary skill in the art, the myosin-1F protein antigen maybe provided by injecting a myosin-1F protein against which antibodiesare desired to be raised into a recipient, or contacting the recipientwith a myosin-1F nucleic acid, capable of expressing the myosin-1Fprotein antigen, under conditions for expression of the myosin-1Fprotein antigen.

[0401] In a preferred embodiment, a therapeutic compound is conjugatedto an antibody, preferably a myosin-1F protein antibody. The therapeuticcompound may be a cytotoxic agent. In this method, targeting the,cytotoxic agent to apoptotic cells or tumor cells results in a reductionin the number of afflicted cells, thereby reducing symptoms associatedwith apoptosis or cancer. Cytotoxic agents are numerous and varied andinclude, but are not limited to, cytotoxic drugs or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies raised against myosin-1F proteins, orbinding of a radionuclide to a chelating agent that has been covalentlyattached to the antibody.

[0402] In a preferred embodiment, myosin-1F protein genes areadministered as DNA vaccines, either single nucleic acids orcombinations of myosin-1F protein genes. Naked DNA vaccines aregenerally known in the art; see Brower, Nature Biotechnology16:1304-1305 (1998). Methods for the use of nucleic acids as DNAvaccines are well known to one of ordinary skill in the art, and includeplacing a myosin-1F protein gene or portion of a myosin-1F proteinnucleic acid under the control of a promoter for expression in apatient. The myosin-1F protein gene used for DNA vaccines can encodefull-length myosin-1F proteins, but more preferably encodes portions ofthe myosin-1F proteins including peptides derived from the myosin-1Fprotein. In a preferred embodiment a patient is immunized with a DNAvaccine comprising a plurality of nucleotide sequences derived from amyosin-1F protein gene. Similarly, it is possible to immunize a patientwith a plurality of myosin-1F protein genes or portions thereof, asdefined herein. Without being bound by theory, following expression ofthe polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helperT-cells and antibodies are induced which recognize and destroy oreliminate cells expressing myosin-1F proteins.

[0403] In a preferred embodiment, the DNA vaccines include a geneencoding an adjuvant molecule with the DNA vaccine. Such adjuvantmolecules include cytokines that increase the immunogenic response tothe myosin-1F protein encoded by the DNA vaccine. Additional oralternative adjuvants are known to those of ordinary skill in the artand find use in the invention.

[0404] The invention also includes the use of myosin-1F proteincompositions, myosin-1F agonists, or myosin-1F antibodies, in thepreparation of a medicament for the treatment of lymphocyte activationdisorders and lymphocyte proliferation disorders.

[0405] All references cited herein are expressly incorporated byreference in their entirety. Moreover, all sequences displayed, cited byreference or accession number in the references are incorporated byreference herein.

We claim:
 1. A method of screening for an immunosuppressant, comprising: a) combining a candidate bioactive agent and a myosin-1F protein; b) detecting modulation in the ATPase activity of said myosin-1F protein in the presence of said candidate bioactive agent; c) contacting said candidate bioactive agent to a B-lymphocyte; d) inducing activation of said lymphocyte; and e) determining the activation of said lymphocyte in the presence of said candidate bioactive agent; wherein said myosin-1F protein exhibits ATPase activity in the absence of said candidate bioactive agent, and wherein a decrease in the activation of said lymphocyte in the presence of said candidate bioactive agent indicates that said candidate bioactive agent is an immunosuppressant.
 2. The method of claim 1, wherein said myosin-1F protein comprises a myosin head domain and an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in SEQ ID NO:2.
 3. The method of claim 2, wherein said myosin-1F protein comprises the amino acid sequence set forth in SEQ ID NO:2.
 4. The method of claim 1, wherein said myosin-1F protein comprises a myosin head domain but lacks a tail domain and consists essentially of an amino acid sequence having at least about 95% identity to the amino acid sequence set forth by amino acids 1-677, 19-677, 1-691, or 12-691 in SEQ ID NO:2.
 5. The method of claim 4, wherein said myosin-1F protein consists essentially of amino acids 1-677, 19-677, 1-691, or 12-691 in SEQ ID NO:2.
 6. A method of screening for an immunosuppressant, comprising: a) combining a candidate bioactive agent, and an myosin-1F protein; b) detecting binding of said myosin-1F protein to said candidate bioactive agent; c) contacting said candidate bioactive agent to a B-lymphocyte; d) inducing activation of said lymphocyte; and e) determining the activation of said lymphocyte in the presence of said candidate bioactive agent; wherein a decrease in the activation of said lymphocyte in the presence of said candidate bioactive agent indicates that said candidate bioactive agent is an immunosuppressant.
 7. The method of claim 6, wherein said myosin-1F protein comprises an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in SEQ ID NO:2.
 8. The method of claim 7, wherein said myosin-1F protein comprises the amino acid sequence set forth in SEQ ID NO:2.
 9. The method of claim 6, wherein said myosin-1F protein comprises a myosin head domain but lacks a tail domain and consists essentially of an amino acid sequence having at least about 95% identity to the amino acid sequence set forth by amino acids 1-677, 19-677, 1-691, or 12-691 in SEQ ID NO:2.
 10. The method of claim 9, wherein said myosin-1F protein consists essentially of amino acids 1-677, 19-677, 1-691, or 12-691 in SEQ ID NO:2.
 11. The method of claim 6, wherein said myosin-1F protein comprises a tail domain but lacks a myosin head domain and consists essentially of an amino acid sequence having at least about 95% identity to the amino acid sequence set forth by amino acids 718-1098 in SEQ ID NO:2.
 12. The method of claim 11, wherein said myosin-1F protein consists essentially of amino acids 718-1098 in SEQ ID NO:2.
 13. The method of claim 6, wherein said myosin-1F protein comprises an IQ domain and a tail domain but lacks a myosin head domain and consists essentially of an amino acid sequence having at least about 95% identity to the amino acid sequence set forth by amino acids 695-1098 in SEQ ID NO:2.
 14. The method of claim 13, wherein said myosin-1F protein consists essentially of amino acids 695-1098 in SEQ ID NO:2.
 15. A method of screening for an immunosuppressant, comprising: a) combining a candidate bioactive agent, a myosin-1F protein, and a myosin-1F binding partner; b) detecting modulation of the binding of said myosin-1F protein to said myosin-1F binding partner in the presence of said candidate bioactive agent; c) contacting said candidate bioactive agent to a B-lymphocyte; d) inducing activation of said lymphocyte; and e) determining the activation of said lymphocyte in the presence of said candidate bioactive agent; wherein said myosin-1F protein will bind to said myosin-1F binding partner in the absence of said candidate bioactive agent, and wherein a decrease in the activation of said lymphocyte in the presence of said candidate bioactive agent indicates that said candidate bioactive agent is an immunosuppressant.
 16. The method of claim 15, wherein said myosin-1F protein comprises an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in SEQ ID NO:2.
 17. The method of claim 16, wherein said myosin-1F protein comprises the amino acid sequence set forth in SEQ ID NO:2.
 18. The method of claim 15, wherein said myosin-1F protein comprises an IQ domain and a tail domain but lacks a myosin head domain and consists essentially of an amino acid sequence having at least about 95% identity to the amino acid sequence set forth by amino acids 695-1098 in SEQ ID NO:2.
 19. The method of claim 18, wherein said myosin-1F protein consists essentially of amino acids 695-1098 in SEQ ID NO:2.
 20. The method of claim 15, wherein said myosin-1F protein comprises a tail domain but lacks a myosin head domain and consists essentially of an amino acid sequence having at least about 95% identity to the amino acid sequence set forth by amino acids 718-1098 in SEQ ID NO:2.
 21. The method of claim 20, wherein said myosin-1F protein consists essentially of amino acids 718-1098 in SEQ ID NO:2.
 22. The method of claim 15, wherein said myosin-1F binding partner is selected from the group consisting of calmodulin, actin, BLNK, ATP, Arp2/3, Bee1p, WASP, WASP-IP, and Vrp1p.
 23. The method of claim 15, wherein said candidate bioactive agent is a small molecule chemical compound from about 200 to about 500 daltons in size.
 24. The method of any of claims 1, 6, and 15, wherein inducing the activation of said B-lymphocyte is done by activating the B cell receptor (BCR).
 25. The method of any of claims 1, 6, and 15, wherein determining the activation of said B-lymphocyte comprises determining the activity of the immunoglobulin heavy chain gene (IgH) promoter.
 26. The method of any of claims 1, 6, and 15, wherein determining the activation of said B-lymphocyte comprises determining the expression of CD69.
 27. The method of any of claims 1, 6, and 15, wherein a library of candidate bioactive agents is combined with said myosin-1F protein.
 28. The method of any of claims 1, 6, and 15, wherein determining the activation of said B-lymphocyte involves sorting said lymphocyte by FACS. 